Secreted salivary zsig63 polypeptide

ABSTRACT

The present invention relates to polynucleotide and polypeptide molecules for zsig63, a novel secreted salivary protein. The polypeptides, and polynucleotides encoding them, may exhibit anti-microbial activity and may be used in the study or treatment of microbial infections. The polynucleotides encoding zsig63, are located on chromosome 4, and can be used to identify a region of the genome associated with human disease states. The present invention also includes antibodies to the zsig63 polypeptides.

REFERENCE TO RELATED APPLICATIONS

[0001] This application is related to Provisional Application No.60/124,820, filed on Mar. 17, 1999. Under 35 U.S.C. § 119(e)(1), thisapplication claims benefit of said Provisional Application.

BACKGROUND OF THE INVENTION

[0002] Bacterial, and other microbial interaction with host tissues canhave beneficial (symbiotic) as well as deleterious (pathogenic)consequences. Invading microbes can be pathogenic. Consequently, hostbiological defense strategies have evolved to protect organisms frominvasion by disease-causing microorganisms.

[0003] Microbial infection response systems include oxidative andnon-oxidative mechanisms, utilizing compounds that are enzymaticallysynthesized in cells, as well as peptides that are single gene products.For example, anti-microbial peptides constitute an oxygen-independenthost defense system found in organisms encompassing many taxonomicfamilies. One major class of anti-microbial peptides is defined byconserved cysteine residue patterns and is termed defensins. Forexample, mammalian defensins, derived from skin, lung and intestine,exhibit antibiotic activity against a wide variety of pathogens,including Gram-positive and Gram-negative bacteria, fungi (e.g., Candidaspecies) and viruses. See, for example, Porter et al., Infect. Immun.65(6): 2396-401, 1997.

[0004] A major class of microbial peptides is called adhesins. Adhesinsenable microbes to adhere to mammalian tissues, for example the oral,gastrointestinal, urogenital and respiratory tracts. For a pathogenicmicroorganism, this may be a primary route to colonization and/orinvasion of the host. Conversely, natural microbial flora can adhere tohost tissues and create beneficial symbiotic relationships such asnutritional benefits, and protection against colonization of pathogenicmicrobes. The host defenses involved in attracting and establishingbeneficial microbial colonization, as opposed to pathogenic microbialcolonization, are not well understood. However, host defenses thataffect this balance may have anti-microbial, immunomodulatory,inflammatory, anti-inflammatory or other properties.

[0005] Thus, moieties having anti-microbial, adhesin-like,immunomodulatory, inflammatory, anti-inflammatory or other propertiesare sought. The present invention provides such polypeptides for theseand other uses that should be apparent to those skilled in the art fromthe teachings herein.

SUMMARY OF THE INVENTION

[0006] Within one aspect, the present invention provides an isolatedpolynucleotide encoding a zsig63 polypeptide comprising a sequence ofamino acid residues that is at least 90% identical to an amino acidsequence selected from the group consisting of: (a) the amino acidsequence as shown in SEQ ID NO: 2 from amino acid number 16 (Arg) toamino acid number 37 (Ser); (b) the amino acid sequence as shown in SEQID NO: 2 from amino acid number 38 (Leu) to amino acid number 126 (Ala);(c) the amino acid sequence as shown in SEQ ID NO: 2 from amino acidnumber 127 (Pro) to amino acid number 219 (Gln); (d) the amino acidsequence as shown in SEQ ID NO:2 from amino acid number 16 (Arg) toamino acid number 219 (Gln); and (e) the amino acid sequence as shown inSEQ ID NO:2 from amino acid number 1 (Met) to amino acid number 219(Gln). In one embodiment, the isolated polynucleotide disclosed aboveencodes a zsig63 polypeptide comprising a sequence of amino acidresidues selected from the group consisting of: (a) the amino acidsequence as shown in SEQ ID NO: 2 from amino acid number 16 (Arg) toamino acid number 37 (Ser); (b) the amino acid sequence as shown in SEQID NO: 2 from amino acid number 38 (Leu) to amino acid number 126 (Ala);(c) the amino acid sequence as shown in SEQ ID NO: 2 from amino acidnumber 127 (Pro) to amino acid number 219 (Gln); (d) the amino acidsequence as shown in SEQ ID NO:2 from amino acid number 16 (Arg) toamino acid number 219 (Gln); and (e) the amino acid sequence as shown inSEQ ID NO:2 from amino acid number 1 (Met) to amino acid number 219(Gln). In another embodiment, the isolated polynucleotide disclosedabove is selected from the group consisting of: (a) a polynucleotidesequence as shown in SEQ ID NO:1 from nucleotide 173 to nucleotide 784;(b) a polynucleotide sequence as shown in SEQ ID NO:1 from nucleotide128 to nucleotide 784; and (c) a polynucleotide sequence complementaryto (a) or (b). In another embodiment, the isolated polynucleotidedisclosed above comprises nucleotide I to nucleotide 657 of SEQ ID NO:3.

[0007] Within another aspect, the present invention provides anexpression vector comprising the following operably linked elements: atranscription promoter; a DNA segment encoding a zsig63 polypeptidecomprising an amino acid sequence that is at least 90% identical to theamino acid sequence shown in SEQ ID NO:2 from amino acid number 16 (Arg)to amino acid number 219 (Gln); and a transcription terminator.

[0008] In one embodiment, the expression vector disclosed above furthercomprises a secretory signal sequence operably linked to the DNAsegment.

[0009] Within another aspect, the present invention provides a culturedcell into which has been introduced an expression vector as disclosedabove, wherein the cell expresses a polypeptide encoded by the DNAsegment.

[0010] Within another aspect, the present invention provides a DNAconstruct encoding a fusion protein, the DNA construct comprising: afirst DNA segment encoding a polypeptide selected from the groupconsisting of: (a) the amino acid sequence of SEQ ID NO: 2 from residuenumber 1 (Met) to residue number 15 (Ala); (b) the amino acid sequenceof SEQ ID NO: 2 from residue number 16 (Arg) to residue number 37 (Ser);(c) the amino acid sequence of SEQ ID NO: 2 from residue number 38 (Leu)to residue number 126 (Ala); (d) the amino acid sequence of SEQ ID NO: 2from residue number 127 (Pro) to residue number 219 (Gln); and (e) theamino acid sequence of SEQ ID NO:2 from residue number 16 (Arg) toresidue number 219 (Gln); and at least one other DNA segment encoding anadditional polypeptide, wherein the first and other DNA segments areconnected in-frame; and encode the fusion protein.

[0011] Within another aspect, the present invention provides a fusionprotein produced by a method comprising: culturing a host cell intowhich has been introduced a vector comprising the following operablylinked elements: (a) a transcriptional promoter; (b) a DNA constructencoding a fusion protein as disclosed above; and(c) a transcriptionalterminator; and recovering the protein encoded by the DNA segment.

[0012] Within another aspect, the present invention provides an isolatedzsig63 polypeptide comprising a sequence of amino acid residues that isat least 90% identical to an amino acid sequence selected from the groupconsisting of: (a) the amino acid sequence as shown in SEQ ID NO: 2 fromamino acid number 16 (Arg) to amino acid number 37 (Ser); (b) the aminoacid sequence as shown in SEQ ID NO: 2 from amino acid number 38 (Leu)to amino acid number 126 (Ala); (c) the amino acid sequence as shown inSEQ ID NO: 2 from amino acid number 127 (Pro) to amino acid number 219(Gln); (d) the amino acid sequence as shown in SEQ ID NO:2 from aminoacid number 16 (Arg) to amino acid number 219 (Gln); and (e) the aminoacid sequence as shown in SEQ ID NO:2 from amino acid number 1 (Met) toamino acid number 219 (Gln). In one embodiment, the isolated polypeptidedisclosed above comprises a sequence of amino acid residues selectedfrom the group consisting of: (a) the amino acid sequence as shown inSEQ ID NO: 2 from amino acid number 16 (Arg) to amino acid number 37(Ser); (b) the amino acid sequence as shown in SEQ ID NO: 2 from aminoacid number 38 (Leu) to amino acid number 126 (Ala); (c) the amino acidsequence as shown in SEQ ID NO: 2 from amino acid number 127 (Pro) toamino acid number 219 (Gln); (d) the amino acid sequence as shown in SEQID NO:2 from amino acid number 16 (Arg) to amino acid number 219 (Gln);and (e) the amino acid sequence as shown in SEQ ID NO:2 from amino acidnumber 1 (Met) to amino acid number 219 (Gln).

[0013] Within another aspect, the present invention provides a method ofproducing a zsig63 polypeptide comprising: culturing a cell as disclosedabove; and isolating the zsig63 polypeptide produced by the cell.

[0014] Within another aspect, the present invention provides a method ofdetecting, in a test sample, the presence of an antagonist of zsig63protein activity, comprising: transfecting a zsig63-responsive cell,with a reporter gene construct that is responsive to a zsig63-stimulatedcellular pathway; and producing a zsig63 polypeptide by the method asdisclosed above; and adding the zsig63 polypeptide to the cell, in thepresence and absence of a test sample; and comparing levels of responseto the zsig63 polypeptide, in the presence and absence of the testsample, by a biological or biochemical assay; and determining from thecomparison, the presence of the antagonist of zsig63 activity in thetest sample.

[0015] Within another aspect, the present invention provides a method ofdetecting, in a test sample, the presence of an agonist of zsig63protein activity, comprising: transfecting a zsig63-responsive cell,with a reporter gene construct that is responsive to a zsig63-stimulatedcellular pathway; and adding a test sample; and comparing levels ofresponse in the presence and absence of the test sample, by a biologicalor biochemical assay; and determining from the comparison, the presenceof the agonist of zsig63 activity in the test sample.

[0016] Within another aspect, the present invention provides a method ofproducing an antibody to zsig63 polypeptide comprising the followingsteps in order: inoculating an animal with a polypeptide selected fromthe group consisting of: (a) a polypeptide consisting of 9 to 204 aminoacids, wherein the polypeptide consists of a contiguous sequence ofamino acids in SEQ ID NO:2 from amino acid number 16 (Ala) to amino acidnumber 219 (Gln); and (b) a polypeptide according as disclosed above;(c) a polypeptide comprising amino acid number 16 (Arg) to 37 (Ser) ofSEQ ID NO:2; (d) a polypeptide comprising amino acid number 38 (Leu) to126 (Ala) of SEQ ID NO:2; (e) a polypeptide comprising amino acid number127 (Pro) to 219 (Gln) of SEQ ID NO:2; (f) a polypeptide comprisingamino acid number 16 (Arg) to amino acid number 219 (Gln) of SEQ IDNO:2; (g) a polypeptide comprising amino acid number 1 (Met) to aminoacid number 219 (Gln) of SEQ ID NO:2; (h) a polypeptide comprising aminoacid number 14 (Phe) to 19 (Arg) of SEQ ID NO:2; (i) a polypeptidecomprising amino acid number 16 (Arg) to 21 (Phe) of SEQ ID NO:2; (j) apolypeptide comprising amino acid number 24 (Gly) to 29 (Asp) of SEQ IDNO:2; (k) a polypeptide comprising amino acid number 25 (Glu) to 30(Asp) of SEQ ID NO:2; (l) a polypeptide comprising amino acid number187Glu) to 192 (Glu) of SEQ ID NO:2; (m) a polypeptide comprising aminoacid number 24 (Gly) to 33 (Pro) of SEQ ID NO:2; (n) a polypeptidecomprising amino acid number 17 (Lys) to 33 (Proy of SEQ ID NO:2; (o) apolypeptide comprising amino acid number 66 (Thr) to 73 (Pro) of SEQ IDNO:2; (p) a polypeptide comprising amino acid number 103 (Pro) to 108(Gly) of SEQ ID NO:2; (q) a polypeptide comprising amino acid number 190(Ala) to 197 (Glu) of SEQ ID NO:2; (r) a polypeptide comprising aminoacid number 202 (Lys) to 215 (Gly) of SEQ ID NO:2; and (s) a polypeptidecomprising amino acid number 190 (Ala) to 215 (Glu) of SEQ ID NO:2; andwherein the polypeptide elicits an immune response in the animal toproduce the antibody; and isolating the antibody from the animal.

[0017] Within another aspect, the present invention provides an antibodyproduced by the method as disclosed above, which binds to a zsig63polypeptide. In one embodiment, the antibody disclosed above theantibody is a monoclonal antibody. Within another aspect, the presentinvention provides an antibody that binds to a polypeptide as disclosedabove.

[0018] These and other aspects of the invention will become evident uponreference to the following detailed description of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0019] Prior to setting forth the invention in detail, it may be helpfulto the understanding thereof to define the following terms.

[0020] The term “affinity tag” is used herein to denote a peptidesegment that can be attached to a polypeptide to provide forpurification of the polypeptide or provide sites for attachment of thepolypeptide to a substrate. In principal, any peptide or protein forwhich an antibody or other specific binding agent is available can beused as an affinity tag. Affinity tags include a poly-histidine tract,protein A (Nilsson et al., EMBO J 4:1075, 1985; Nilsson et al., MethodsEnzymol. 198:3, 1991), glutathione S transferase (Smith and Johnson,Gene 67:31, 1988), substance P, Flag™ peptide (Hopp et al.,Biotechnology 6:1204-1210, 1988; available from Eastman Kodak Co., NewHaven, Conn.), streptavidin binding peptide, or other antigenic epitopeor binding domain. See, in general Ford et al., Protein Expression andPurification 2:95-107, 1991. DNAs encoding affinity tags are availablefrom commercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).

[0021] The term “allelic variant” denotes any of two or more alternativeforms of a gene occupying the same chromosomal locus. Allelic variationarises naturally through mutation, and may result in phenotypicpolymorphism within populations.

[0022] Gene mutations can be silent (no change in the encodedpolypeptide) or may encode polypeptides having altered amino acidsequence. The term allelic variant is also used herein to denote aprotein encoded by an allelic variant of a gene.

[0023] The terms “amino-terminal” and “carboxyl-terminal” are usedherein to denote positions within polypeptides and proteins. Where thecontext allows, these terms are used with reference to a particularsequence or portion of a polypeptide or protein to denote proximity orrelative position. For example, a certain sequence positionedcarb6xyl-terminal to a reference sequence within a protein is locatedproximal to the carboxyl terminus of the reference sequence, but is notnecessarily at the carboxyl terminus of the complete protein.

[0024] The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions.

[0025] For instance, biotin and avidin (or streptavidin) areprototypical members of a complement/anti-complement pair. Otherexemplary complement/anti-complement pairs include receptor/ligandpairs, antibody/antigen (or hapten or epitope) pairs, sense/antisensepolynucleotide pairs, and the like. Where subsequent dissociation of thecomplement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity of<10⁹ M⁻¹.

[0026] The term “complements of polynucleotide molecules” denotespolynucleotide molecules having a complementary base sequence andreverse orientation as compared to a reference sequence. For example,the sequence 5′ ATGCACGGG 3′ is complementary to 5′ C.CCGTGCAT 3′.

[0027] The term “contig” denotes a polynucleotide that has a contiguousstretch of identical or complementary sequence to anotherpolynucleotide. Contiguous sequences are said to “overlap” a givenstretch of polynucleotide sequence either in their entirety or along apartial stretch of the polynucleotide. For example, representativecontigs to the polynucleotide sequence 5′-ATGGAGCTT-3′ are5′AGCTTgagt-3′ and 3′-tcgacTACC-5′.

[0028] The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

[0029] The term “expression vector” denotes a DNA molecule, linear orcircular, that comprises a segment encoding a polypeptide of interestoperably linked to additional segments that provide for itstranscription. Such additional segments may include promoter andterminator sequences, and may optionally include one or more origins ofreplication, one or more selectable markers, an enhancer, apolyadenylation signal, and the like. Expression vectors are generallyderived from plasmid or viral DNA, or may contain elements of both.

[0030] The term “isolated”, when applied to a polynucleotide molecule,denotes that the polynucleotide has been removed from its naturalgenetic milieu and is thus free of other extraneous or unwanted codingsequences, and is in a form suitable for use within geneticallyengineered protein production systems. Such isolated molecules are thosethat are separated from their natural environment and include cDNA andgenomic clones. Isolated DNA molecules of the present invention are freeof other genes with which they are ordinarily associated, but mayinclude naturally occurring 5′ and 3′ untranslated regions such aspromoters and terminators. The identification of associated regions willbe evident to one of ordinary skill in the art (see for example, Dynanand Tijan, Nature 316:774-78, 1985). When applied to a protein, the term“isolated” indicates that the protein is found in a condition other thanits native environment, such as apart from blood and animal tissue. In apreferred form, the isolated protein is substantially free of otherproteins, particularly other proteins of animal origin. It is preferredto provide the protein in a highly purified form, i.e., greater than 95%pure, more preferably greater than 99% pure.

[0031] The term “operably linked”, when referring to DNA segments,denotes that the segments are arranged so that they function in concertfor their intended purposes, e.g. transcription initiates in thepromoter and proceeds through the coding segment to the terminator.

[0032] The term “ortholog” denotes a polypeptide or protein obtainedfrom one species that is the functional counterpart of a polypeptide orprotein from a different species. Sequence differences among orthologsare the result of speciation. “Paralogs” are distinct but structurallyrelated proteins made by an organism. Paralogs are believed to arisethrough gene duplication. For example, α-globin, β-globin, and myoglobinare paralogs of each other.

[0033] The term “polynucleotide” denotes a single- or double-strandedpolymer of deoxyribonucleotide or ribonucleotide bases read from the 5′to the 3′ end.

[0034] Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules.

[0035] Sizes of polynucleotides are expressed as base pairs (abbreviated“bp”), nucleotides (“nt”), or kilobases (“kb”). Where the contextallows, the latter two terms may describe polynucleotides that aresingle-stranded or double-stranded. When the term is applied todouble-stranded molecules it is used to denote overall length and willbe understood to be equivalent to the term “base pairs”. It will berecognized by those skilled in the art that the two strands of adouble-stranded polynucleotide may differ slightly in length and thatthe ends thereof may be staggered as a result of enzymatic cleavage;thus all nucleotides within a double-stranded polynucleotide moleculemay not be paired.

[0036] A “polypeptide” is a polymer of amino acid residues joined bypeptide bonds, whether produced naturally or synthetically. Polypeptidesof less than about 10 amino acid residues are commonly referred to as“peptides”.

[0037] The term “promoter” denotes a portion of a gene containing DNAsequences that provide for the binding of RNA polymerase and initiationof transcription. Promoter sequences are commonly, but not always, foundin the 5′ non-coding regions of genes.

[0038] The term “receptor” denotes a cell-associated protein that bindsto a bioactive molecule (i.e., a ligand) and mediates the effect of theligand on the cell. Membrane-bound receptors are characterized by amulti-domain structure comprising an extracellular ligand-binding domainand an intracellular effector domain that is typically involved insignal transduction. Binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell. This interactionin turn leads to an alteration in the metabolism of the cell. Metabolicevents that are linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids. Most nuclear receptors also exhibit amulti-domain structure, including an amino-terminal, transactivatingdomain, a DNA binding domain and a ligand-binding domain. In general,receptors can be membrane bound, cytosolic or nuclear; monomeric (e.g.,thyroid stimulating hormone receptor, beta-adrenergic receptor) ormultimeric (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor,GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6receptor).

[0039] The term “secretory signal sequence” denotes a DNA sequence thatencodes a polypeptide (a “secretory peptide”) that, as a component of alarger polypeptide, directs the larger polypeptide through a secretorypathway of a cell in which it is synthesized. The larger peptide iscommonly cleaved to remove the secretory peptide during transit throughthe secretory pathway.

[0040] A “soluble receptor” is a receptor polypeptide that is not boundto a cell membrane. Soluble receptors are most commonly ligand-bindingreceptor polypeptides that lack transmembrane and cytoplasmic domains.Soluble receptors can comprise additional amino acid residues, such asaffinity tags that provide for purification of the polypeptide orprovide sites for attachment of the polypeptide to a substrate, orimmunoglobulin constant region sequences. Many cell-surface receptorshave naturally occurring, soluble counterparts that are produced byproteolysis or translated from alternatively spliced mRNAs. Receptorpolypeptides are said to be substantially free of transmembrane andintracellular polypeptide segments when they lack sufficient portions ofthese segments to provide membrane anchoring or signal transduction,respectively.

[0041] Molecular weights and lengths of polymers determined by impreciseanalytical methods (e.g., gel electrophoresis) will be understood to beapproximate values. When such a value is expressed as “about” X or“approximately” X, the stated value of X will be understood to beaccurate to ±10%.

[0042] All references cited herein are incorporated by reference intheir entirety.

[0043] The present invention is based in part upon the discovery of anovel DNA sequence that encodes a human polypeptide having structuralsimilarity to proteins of the bacterial adhesin family.

[0044] A standard Northern blot tissue distribution of the mRNAcorresponding to this novel DNA revealed high expression in salivarygland, and moderate to high expression in thyroid. Such high expressionin salivary gland is consistent with the knowledge in the art regardinganti-bacterial polypeptides, such as defensins, i.e., that they areexpressed in epithelial tissues, and are highly inducible upon microbialinfection.

[0045] The novel zsig63 polypeptides of the present invention wereinitially identified by querying an EST database for proteins homologousto proteins having a secretory signal sequence. These proteins arecharacterized by an upstream methionine start site and a hydrophobicregion of approximately 13 amino acids, followed by a peptide signalpeptidase cleavage site. An EST database was queried for novel DNAsequences whose translations would meet these search criteria. An ESTwas found and its corresponding cDNA was sequenced. The zsig63nucleotide sequence is believed to encode the entire coding sequence ofthe predicted protein. Zsig63 may be a novel host-defense polypeptide,immune modulating factor, anti-pathogenic polypeptide, cell-cellsignaling molecule, growth factor, cytokine, secreted extracellularmatrix associated protein with growth factor hormone activity, or thelike, and is a member a novel protein family.

[0046] The sequence of the zsig63 polypeptide was obtained from a singleclone believed to contain its corresponding polynucleotide sequence. Theclone was obtained from a salivary gland library. Other libraries thatmight also be searched for such sequences include thyroid, prostate, andthe like.

[0047] The nucleotide sequence of a representative zsig63-encoding DNAis described in SEQ ID NO:1 (from nucleotide 128 to 784), and itsdeduced 219 amino acid sequence is described in SEQ ID NO:2. In itsentirety, the zsig63 polypeptide (SEQ ID NO:2) represents a full-lengthpolypeptide segment (residue 1 (Met) to residue 219 (Gln) of SEQ IDNO:2). The domains and structural features of the zsig63 polypeptide arefurther described below.

[0048] Analysis of the zsig63 polypeptide encoded by the DNA sequence ofSEQ ID NO:1 revealed an open reading frame encoding 219 amino acids (SEQID NO:2) comprising a predicted secretory signal peptide of 15 aminoacid residues (residue 1 (Met) to residue 15 (Ala) of SEQ ID NO:2), anda mature polypeptide of 204 amino acids (residue 16 (Arg) to residue 219(Gln) of SEQ ID NO:2). Structural analysis revealed the following 3structural domains:

[0049] (1) The first domain, referred to hereinafter as “domain 1,”corresponds approximately to amino acid residues 16 (Arg) to amino acidresidue 37 (Ser) of SEQ ID NO:2. Domain 1 contains an acid region of 5acid residues (corresponding to amino acid residues 25 (Glu) to aminoacid residue 30 (Asp) of SEQ ID NO:2).

[0050] (2) The second domain, referred to hereinafter as “domain 2,”corresponds approximately to amino acid residues 38 (Leu) to amino acidresidue 126 (Ala) of SEQ ID NO:2. Domain 2 contains a high concentrationof Tyrosine residues (16% over 49 amino acids). Tyrosine residues indomain 2 are present, in reference to SEQ ID NO:2 at the followingpositions: 42, 52, 53, 62, 67, 75, 86, and 88.

[0051] (3) The third domain, referred to hereinafter as “domain 3,”corresponds approximately to amino acid residues 127 (Pro) to amino acidresidue 219 (Gln) of SEQ ID NO:2. Domain 3 contains a region rich incoil-like structure (corresponding to amino acid residues 127 (Pro) toamino acid residue 208 (Pro) of SEQ ID NO:2) that contains 16 fullevenly-spaced coil-like repeats punctuated by proline residues every 5amino acids. These repeats are defined as follows, based on the prolineresidues and corresponding reference to SEQ ID NO:2:

[0052] “repeat 1” corresponding to amino acid residues 124 (Ile) toamino acid residue 133 (Pro) of SEQ ID NO:2;

[0053] “repeat 2” corresponding to amino acid residues 134 (Ala) toamino acid residue 138 (Pro) of SEQ ID NO:2;

[0054] “repeat 3” corresponding to amino acid residues 139 (Leu) toamino acid residue 143 (Pro) of SEQ ID NO:2;

[0055] “repeat 4” corresponding to amino acid residues 144 (Val) toamino acid residue 148 (Pro) of SEQ ID NO:2;

[0056] “repeat 5” corresponding to amino acid residues 149 (Ala) toamino acid residue 153 (Pro) of SEQ ID NO:2;

[0057] “repeat 6” corresponding to amino acid residues 154 (Val) toamino acid residue 158 (Pro) of SEQ ID NO:2.

[0058] “repeat 7” corresponding to amino acid residues 159 (Ala) toamino acid residue 163 (Pro) of SEQ ID NO:2;

[0059] “repeat 8” corresponding to amino acid residues 164 (Val) toamino acid residue 168 (Pro) of SEQ ID NO:2;

[0060] “repeat 9” corresponding to amino acid residues 169 (Ala) toamino acid residue 173 (Pro) of SEQ IDNO:2;

[0061] “repeat 10” corresponding to amino acid residues 174 (Val) toamino acid residue 178 (Pro) of SEQ ID NO:2;

[0062] “repeat 11” corresponding to amino acid residues 179 (Ala) toamino acid residue 183 (Pro) of SEQ ID NO:2;

[0063] “repeat 12” corresponding to amino acid residues 184 (Val) toamino acid residue 188 (Pro) of SEQ ID NO:2.

[0064] “repeat 13” corresponding to amino acid residues 189 (Ala) toamino acid residue 193 (Pro) of SEQ ID NO:2;

[0065] “repeat 14” corresponding to amino acid residues 194 (Ser) toamino acid residue 198 (Pro) of SEQ ID NO:2;

[0066] “repeat 15” corresponding to amino acid residues 199 (Ala) toamino acid residue 203 (Pro) of SEQ ID NO:2; and

[0067] “repeat 16” corresponding to amino acid residues 204 (Ala) toamino acid residue 208 (Pro) of SEQ ID NO:2.

[0068] Structural modeling is performed by one of skill in the art,using modelling software, such as InsightII®(Biosym/MSI, San Diego,Calif.). The presence of low variance and structural motifs generallycorrelate with or define important structural regions in proteins.Regions of low variance (e.g., hydrophobic clusters) are generallypresent in regions of structural importance (Sheppard, P. et al.,supra.). Such regions of low variance often contain rare or infrequentamino acids, such as Tryptophan. The regions flanking and between suchconserved and low variance motifs may be more variable, but are oftenfunctionally significant because they may relate to or define importantstructures and activities such as binding domains, biological andenzymatic activity, signal transduction, cell-cell interaction, tissuelocalization domains and the like.

[0069] The coil-like repeats in domain 3 of zsig63 polypeptide can beused as a tool to identify new family members. For instance, reversetranscription-polymerase chain reaction (RT-PCR) can be used to amplifysequences encoding the conserved motif from RNA obtained from a varietyof tissue sources. More specifically, degenerate DNA probes anddegenerate primers can be employed to identify other human orzsig63-like polynucleotides. In particular, highly degenerate primersdesigned from the above sequences are useful for this purpose.

[0070] SEQ ID NO:3 is a degenerate polynucleotide sequence thatencompasses all polynucleotides that encode the zsig63 polypeptide ofSEQ ID NO:2. Thus, zsig63 polypeptide-encoding polynucleotides rangingfrom nucleotide 1 to nucleotide 657 of SEQ ID NO:3 are contemplated bythe present invention. Also contemplated by the present invention arefragments and fusions as described above with respect to SEQ ID NO:1 andwhich are formed from analogous regions of SEQ ID NO:3. The symbols inSEQ ID NO:3 are summarized in Table 1 below. TABLE 1 NucleotideResolutions Complement Resolutions A A T T C C G G G G C C T T A A R A|GY C|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G S A|T W A|T W C|G HA|C|T D A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|T H A|C|T N A|C|G|TN A|C|G|T

[0071] TABLE 2 Amino Degenerate Acid Letter Codons Codon Cys C TGC TGTTGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT ACN Pro PCCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGNAsn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q CAA CAG CARHis H CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys K AAA AAG AARMet M ATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTG YTNVal V GTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr Y TAC TAT TAY Trp W TGGTGG Ter . TAA TAG TGA TRR Asn|Asp B RAY Glu|Gln Z SAR Any X NNN Gap — —

[0072] One of ordinary skill in the art will appreciate that someambiguity is introduced in determining a degenerate codon,representative of all possible codons encoding each amino acid. Forexample, the degenerate codon for serine (WSN) can, in somecircumstances, encode arginine (AGR); and the degenerate codon forarginine (MGN) can, in some circumstances, encode serine (AGY). Asimilar relationship exists between codons encoding phenylalanine andleucine. Thus, some polynucleotides encompassed by the degeneratesequence may encode variant amino acid sequences, but one of ordinaryskill in the art can easily identify such variant sequences by referenceto the amino acid sequence of SEQ ID NO:2. Variant sequences can bereadily tested for functionality as described herein.

[0073] One of ordinary skill in the art will also appreciate thatdifferent species can exhibit “preferential codon usage.” In general,see, Grantham, et al., Nuc. Acids Res. 8:1893-912, 1980; Haas, et al.Curr. Biol. 6:315-24, 1996; Wain-Hobson, et al., Gene 13:355-64, 1981;Grosjean and Fiers, Gene 18:199-209, 1982; Holm, Nuc. Acids Res.14:3075-87, 1986; Ikemura, J. Mol. Biol. 158:573-97, 1982. As usedherein, the term “preferential codon usage” or “preferential codons” isa term of art referring to protein translation codons that are mostfrequently used in cells of a certain species, thus favoring one or afew representatives of the possible codons encoding each amino acid (SeeTable 2). For example, the amino acid Threonine (Thr) may be encoded byACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonlyused codon; in other species, for example, insect cells, yeast, virusesor bacteria, different Thr codons may be preferential. Preferentialcodons for a particular species can be introduced into thepolynucleotides of the present invention by a variety of methods knownin the art. Introduction of preferential codon sequences intorecombinant DNA can, for example, enhance production of the protein bymaking protein translation more efficient within a particular cell typeor species. Therefore, the degenerate codon sequence disclosed in SEQ IDNO:3 serves as a template for optimizing expression of polynucleotidesin various cell types and species commonly used in the art and disclosedherein. Sequences containing preferential codons can be tested andoptimized for expression in various species, and tested forfunctionality as disclosed herein.

[0074] Within preferred embodiments of the invention the isolatedpolynucleotides will hybridize to similar sized regions of SEQ ID NO: 1,or a sequence complementary thereto, under stringent conditions. Ingeneral, stringent conditions are selected to be about 5° C. lower thanthe thermal melting point (T_(m)) for the specific sequence at a definedionic strength and pH. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of the target sequence hybridizes to aperfectly matched probe. Numerous equations for calculating T_(m) areknown in the art, and are specific for DNA, RNA and DNA-RNA hybrids andpolynucleotide probe sequences of varying length (see, for example,Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition(Cold Spring Harbor Press 1989); Ausubel et al., (eds.), CurrentProtocols in Molecular Biology (John Wiley and Sons, Inc. 1987); Bergerand Kimmel (eds.), Guide to Molecular Cloning Techniques, (AcademicPress, Inc. 1987); and Wetmur, Crit. Rev. Biochem. Mol. Biol. 26:227(1990)). Sequence analysis software such as OLIGO 6.0 (LSR; Long Lake,Minn.) and Primer Premier 4.0™ (Premier Biosoft International; PaloAlto, Calif.), as well as sites on the Internet, are available tools foranalyzing a given sequence and calculating T^(m) based on user definedcriteria. Such programs can also analyze a given sequence under definedconditions and identify suitable probe sequences. Typically,hybridization of longer polynucleotide sequences, >50 base pairs, isperformed at temperatures of about 20-25° C. below the calculated T_(m).For smaller probes, <50 base pairs, hybridization is typically carriedout at the T_(m) or 5-10° C. below. This allows for the maximum rate ofhybridization for DNA-DNA and DNA-RNA hybrids. Higher degrees ofstringency at lower temperatures can be achieved with the addition offormamide which reduces the T_(m) of the hybrid about 1° C. for each 1%formamide in the buffer solution. Suitable stringent hybridizationconditions are equivalent to about a 5 h to overnight incubation atabout 42° C. in a solution comprising: about 40-50% formamide, up toabout 6X SSC, about 5X Denhardt's solution, zero up to about 10% dextransulfate, and about 10-20 μg/ml denatured commercially-available carrierDNA. Generally, such stringent conditions include temperatures of 20-70°C. and a hybridization buffer containing up to 6x SSC and 0-50%formamide; hybridization is then followed by washing filters in up toabout 2X SSC. For example, a suitable wash stringency is equivalent to0.1X SSC to 2X SSC, 0.1% SDS, at 55° C. to 65° C. Different degrees ofstringency can be used during hybridization and washing to achievemaximum specific binding to the target sequence. Typically, the washesfollowing hybridization are performed at increasing degrees ofstringency to remove non-hybridized polynucleotide probes fromhybridized complexes. Stringent hybridization and wash conditions dependon the length of the probe, reflected in the Tm, hybridization and washsolutions used, and are routinely determined empirically by one of skillin the art.

[0075] As previously noted, the isolated polynucleotides of the presentinvention include DNA and RNA. Methods for isolating DNA and RNA arewell known in the art. It is generally preferred to isolate RNA frombronchial epithelium, although DNA can also be prepared using RNA fromother tissues or isolated as genomic DNA. Total RNA can be preparedusing guanidine HCL extraction followed by isolation by centrifugationin a CsCl gradient (Chirgwin et al., Biochemistry 18:52-94, 1979). Poly(A)⁺RNA is prepared from total RNA using the method of Aviv and Leder(Proc. Natl. Acad. Sci. USA 69:1408-1412, 1972). Complementary DNA(cDNA) is prepared from poly(A)₊RNA using known methods. Polynucleotidesencoding zsig63 polypeptides are then identified and isolated by, forexample, hybridization or PCR.

[0076] The present invention further provides counterpart polypeptidesand polynucleotides from other species (orthologs). These speciesinclude, but are not limited to mammalian, avian, amphibian, reptile,fish, insect and other vertebrate and invertebrate species. Ofparticular interest are zsig63 polypeptides from other mammalianspecies, including murine, rat, porcine, ovine, bovine, canine, feline,equine and other primate proteins. Species homologs of the humanproteins can be cloned using information and compositions provided bythe present invention in combination with conventional cloningtechniques. For example, a cDNA can be cloned using mRNA obtained from atissue or cell type that expresses the protein. Suitable sources of mRNAcan be identified by probing Northern blots with probes designed fromthe sequences disclosed herein. A library is then prepared from mRNA ofa positive tissue of cell line. A zsig63 polypeptide-encoding cDNA canthen be isolated by a variety of methods, such as by probing with acomplete or partial human cDNA or with one or more sets of degenerateprobes based on the disclosed sequences. A CDNA can also be cloned usingthe polymerase chain reaction, or PCR (Mullis, U.S. Pat. No. 4,683,202),using primers designed from the sequences disclosed herein. Within anadditional method, the cDNA library can be used to transform ortransfect host cells, and expression of the cDNA of interest can bedetected with an antibody to zsig63 polypeptide. Similar techniques canalso be applied to the isolation of genomic clones.

[0077] Those skilled in the art will recognize that the sequencesdisclosed in SEQ ID NO:1 and SEQ ID NO:2 represent a single allele ofthe human zsig63 gene and polypeptide, and that allelic variation andalternative splicing are expected to occur. Allelic variants can becloned by probing cDNA or genomic libraries from different individualsaccording to standard procedures. Allelic variants of the DNA sequenceshown in SEQ ID NO:2 including those containing silent mutations andthose in which mutations result in amino acid sequence changes, arewithin the scope of the present invention.

[0078] The present invention also provides isolated zsig63 polypeptidesthat are substantially similar to the polypeptides of SEQ ID NO:2 andtheir orthologs. The term “substantially similar” is used herein todenote polypeptides having 60%, preferably 70% and more preferably atleast 80%, sequence identity to the sequences shown in SEQ ID NO:2 ortheir orthologs or paralogs. Such polypeptides will more preferably beat least 90% identical, and most preferably 95% or more identical to SEQID NO:2 or its orthologs or paralogs. Percent sequence identity isdetermined by conventional methods. See, for example, Altschul et al.,Bull. Math. Bio. 48:603-616, 1986 and Henikoff and Henikoff, Proc. Natl.Acad. Sci. USA 89:10915-10919, 1992. Briefly, two amino acid sequencesare aligned to optimize the alignment scores using a gap opening penaltyof 10, a gap extension penalty of 1, and the “blosum 62” scoring matrixof Henikoff and Henikoff (ibid.) as shown in Table 3 (amino acids areindicated by the standard one-letter codes). The percent identity isthen calculated as:$\frac{{Total}\quad {number}\quad {of}\quad {identical}\quad {matches}}{\begin{matrix}\lbrack {{length}\quad {of}\quad {the}\quad {longer}\quad {sequence}\quad {plus}}  \\{{number}\quad {of}\quad {gaps}\quad {introduced}\quad {into}\quad {the}} \\{{longer}\quad {sequence}\quad {in}\quad {order}\quad {to}\quad {align}\quad {the}} \\ {{two}\quad {sequence}} \rbrack\end{matrix}} \times 100$

TABLE 3 A R N D C Q E G H I L K M F P S T W Y V A 4 R −1 5 N −2 0 6 D −2−2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 2 5 G 0 −2 0 −1 −3−2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3 −4 −3 4 L −1 −2−3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 M −1 −1 −2 −3−1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6 P −1 −2−2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0 −1 −2 −2 0 −1−2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W −3 −3 −4 −4−2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1 −2 −3 2 −1−1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1 −1 −2 −2 0−3 −1 4

[0079] Sequence identity of polynucleotide molecules is determined bysimilar methods using a ratio as disclosed above.

[0080] Those skilled in the art appreciate that there are manyestablished algorithms available to align two amino acid sequences. The“FASTA” similarity search algorithm of Pearson and Lipman is a suitableprotein alignment method for examining the level of identity shared byan amino acid sequence disclosed herein and the amino acid sequence of aputative variant zsig63. The FASTA algorithm is described by Pearson andLipman, Proc. Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth.Enzymol. 183:63 (1990).

[0081] Briefly, FASTA first characterizes sequence similarity byidentifying regions shared by the query sequence (e.g., SEQ ID NO:2) anda test sequence that have either the highest density of identities (ifthe ktup variable is 1) or pairs of identities (if ktup=2), withoutconsidering conservative amino acid substitutions, insertions, ordeletions. The ten regions with the highest density of identities arethen rescored by comparing the similarity of all paired amino acidsusing an amino acid substitution matrix, and the ends of the regions are“trimmed” to include only those residues that contribute to the highestscore. If there are several regions with scores greater than the“cutoff” value (calculated by a predetermined formula based upon thelength of the sequence and the ktup value), then the trimmed initialregions are examined to determine whether the regions can be joined toform an approximate alignment with gaps. Finally, the highest scoringregions of the two amino acid sequences are aligned using a modificationof the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. MolBiol. 48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), whichallows for amino acid insertions and deletions. Illustrative parametersfor FASTA analysis are: ktup=1, gap opening penalty=10, gap extensionpenalty=1, and substitution matrix=BLOSUM62. These parameters can beintroduced into a FASTA program by modifying the scoring matrix file(“SMATRIX”), as explained in Appendix 2 of Pearson, Meth. Enzymol.183:63 (1990).

[0082] FASTA can also be used to determine the sequence identity ofnucleic acid molecules using a ratio as disclosed above. For nucleotidesequence comparisons, the ktup value can range between one to six,preferably from four to six.

[0083] The BLOSUM62 table (Table 3) is an amino acid substitution matrixderived from about 2,000 local multiple alignments of protein sequencesegments, representing highly conserved regions of more than 500 groupsof related proteins (Henikoff and Henikoff, Proc. Nat'l Acad. Sci. USA89:10915 (1992)). Accordingly, the BLOSUM62 substitution frequencies canbe used to define conservative amino acid substitutions that may beintroduced into the amino acid sequences of the present invention.Although it is possible to design amino acid substitutions based solelyupon chemical properties (as discussed below), the language“conservative amino acid substitution” preferably refers to asubstitution represented by a BLOSUM62 value of greater than -1. Forexample, an amino acid substitution is conservative if the substitutionis characterized by a BLOSUM62 value of 0, 1, 2, or 3. According to thissystem, preferred conservative amino acid substitutions arecharacterized by a BLOSUM62 value of at least 1 (e.g., 1, 2 or 3), whilemore preferred conservative amino acid substitutions are characterizedby a BLOSUM62 value of at least 2 (e.g., 2 or 3).

[0084] Variant proteins and polypeptides are characterized as having oneor more amino acid substitutions, deletions or additions. These changesare preferably of a minor nature, that is conservative amino acidsubstitutions (see Table 4) and other substitutions that do notsignificantly affect the folding or activity of the protein orpolypeptide; small deletions, typically of one to about 30 amino acids;and small amino- or carboxyl-terminal extensions, such as anamino-terminal methionine residue, a small linker peptide of up to about20-25 residues, or a small extension that facilitates purification (anaffinity tag), such as a poly-histidine tract, protein A (Nilsson etal., EMBO J. 4:1075, 1985; Nilsson et al., Methods Enzymol. 198:3,1991), glutathione S transferase (Smith and Johnson, Gene 67:31, 1988),maltose binding protein (Kellerman and Ferenci, Methods Enzymol.90:459-463, 1982; Guan et al., Gene 67:21-30, 1987), thioredoxin,ubiquitin, cellulose binding protein, T7 polymerase, or other antigenicepitope or binding domain. See, in general Ford et al., ProteinExpression and Purification 2:95-107, 1991. DNAs encoding affinity tagsare available from commercial suppliers (e.g., Pharmacia Biotech,Piscataway, N.J.; New England Biolabs, Beverly, Mass.). The presentinvention thus includes polypeptides of from about 170 to about 250amino acid residues that comprise a sequence that is at least 80%,preferably at least 90%, and more preferably 95% or more identical tothe corresponding region of SEQ ID NO:2. Polypeptides comprisingaffinity tags can further comprise a proteolytic cleavage site betweenthe zsig63 polypeptide and the affinity tag. Preferred such sitesinclude thrombin cleavage sites and factor Xa cleavage sites. TABLE 4Conservative amino acid substitutions Basic: arginine lysine histidineAcidic: glutamic acid aspartic acid Polar: glutamine asparagineHydrophobic: leucine isoleucine valine Aromatic: phenylalaninetryptophan tyrosine Small: glycine alanine serine threonine methionine

[0085] The proteins of the present invention can also comprise, inaddition to the 20 standard amino acids, non-naturally occurring aminoacid residues. Non-naturally occurring amino acids include, withoutlimitation, trans-3-methylproline,

[0086]2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline,N-methyl-glycine, allo-threonine, methylthreonine,hydroxyethyl-cysteine, hydroxyethylhomocysteine, nitroglutamine,homoglutamine, pipecolici acid, tert-leucine, norvaline,2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenyl-alanine,4-fluorophenylalanine, 4-hydroxyproline, 6-N-methyl lysine,2-aminoisobutyric acid, isovaline and a-methyl serine. Several methodsare known in the art for incorporating non-naturally occurring aminoacid residues into proteins. For example, an in vitro system can beemployed wherein nonsense mutations are suppressed using chemicallyaminoacylated suppressor tRNAs. Methods for synthesizing amino acids andaminoacylating tRNA are known in the art. Transcription and translationof plasmids containing nonsense mutations are carried out in a cell freesystem comprising an E. coli S30 extract and commercially availableenzymes and other reagents. Proteins are purified by chromatography.See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991;Ellman et al., Meth. Enzymol. 202:301, 1991; Chung et al., Science259:806-09, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA90:10145-49, 1993). In a second method, translation is carried out inXenopus oocytes by microinjection of mutated mRNA and chemicallyaminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem.271:19991-98, 1996). Within a third method, E. coli cells are culturedin the absence of a natural amino acid that is to be replaced (e.g.,phenylalanine) and in the presence of the desired non-naturallyoccurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturallyoccurring amino acid is incorporated into the protein in place of itsnatural counterpart. See, Koide et al., Biochem. 33:7470-76, 1994.Naturally occurring amino acid residues can be converted tonon-naturally occurring species by in vitro chemical modification.Chemical modification can be combined with site-directed mutagenesis tofurther expand the range of substitutions (Wynn and Richards, ProteinSci. 2:395-403, 1993).

[0087] A limited number of non-conservative amino acids, amino acidsthat are not encoded by the genetic code, non-naturally occurring aminoacids, and unnatural amino acids may be substituted for zsig63polypeptide amino acid residues. “Unnatural amino acids” have beenmodified after protein synthesis, and/or have a chemical structure intheir side chain(s) different from that of the standard amino acids.Unnatural amino acids can be chemically synthesized, or preferably, arecommercially available, and include pipecolic acid, thiazolidinecarboxylic acid, dehydroproline, 3- and 4-methylproline, and3,3-dimethylproline.

[0088] Essential amino acids in the zsig63 polypeptides of the presentinvention can be identified according to procedures known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244:1081-1085, 1989). In the lattertechnique, single alanine mutations are introduced at every residue inthe molecule, and the resultant mutant molecules are tested forbiological activity (e.g., anti-microbial activity) to identify aminoacid residues that are critical to the activity of the molecule. Seealso, Hilton et al., J. Biol. Chem. 271:4699-4708, 1996. Sites ofligand-receptor or other biological interaction can also be determinedby physical analysis of structure, as determined by such techniques asnuclear magnetic resonance, crystallography, electron diffraction orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids; See, for example, de Vos et al., Science 255:306-312,1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al.,FEBS Lett. 309:59-64, 1992. The identities of essential amino acids canalso be inferred from analysis of homologies with related β-defensins.

[0089] Determination of amino acid residues that are within regions ordomains that are critical to maintaining structural integrity can bedetermined. Within these regions one can determine specific residuesthat will be more or less tolerant of change and maintain the overalltertiary structure of the molecule. Methods for analyzing sequencestructure include, but are not limited to, alignment of multiplesequences with high amino acid or nucleotide identity and computeranalysis using available software (e.g., the Insight II® viewer andhomology modeling tools; MSI, San Diego, Calif.), secondary structurepropensities, binary patterns, complementary packing and buried polarinteractions (Barton, Current Opin. Struct. Biol. 5:372-376, 1995 andCordes et al., Current Opin. Struct. Biol. 6:3-10, 1996). In general,when designing modifications to molecules or identifying specificfragments determination of structure will be accompanied by evaluatingactivity of modified molecules.

[0090] Amino acid sequence changes are made in zsig63 polypeptides so asto minimize disruption of higher order structure essential to biologicalactivity. For example, when the zsig63 polypeptide comprises one or morehelices, changes in amino acid residues will be made so as not todisrupt the helix geometry and other components of the molecule wherechanges in conformation abate some critical function, for example,binding of the molecule to its binding partners. The effects of aminoacid sequence changes can be predicted by, for example, computermodeling as disclosed above or determined by analysis of crystalstructure (see, e.g., Lapthorn et al., Nat. Struct. Biol. 2:266-268,1995). Other techniques that are well known in the art compare foldingof a variant protein to a standard molecule (e.g., the native protein).For example, comparison of the cysteine pattern in a variant andstandard molecules can be made. Mass spectrometry and chemicalmodification using reduction and alkylation provide methods fordetermining cysteine residues which are associated with disulfide bondsor are free of such associations (Bean et al., Anal. Biochem.201:216-226, 1992; Gray, Protein Sci. 2:1732-1748, 1993; and Pattersonet al., Anal. Chem. 66:3727-3732, 1994). It is generally believed thatif a modified molecule does not have the same disulfide bonding patternas the standard molecule folding would be affected. Another well knownand accepted method for measuring folding is circular dichrosism (CD).Measuring and comparing the CD spectra generated by a modified moleculeand standard molecule is routine (Johnson, Proteins 7:205-214, 1990).Crystallography is another well known method for analyzing folding andstructure. Nuclear magnetic resonance (NMR), digestive peptide mappingand epitope mapping are also known methods for analyzing folding andstructural similarities between proteins and polypeptides (Schaanan etal., Science 257:961-964, 1992).

[0091] A Hopp/Woods hydrophilicity profile of the zsig63 proteinsequence as shown in SEQ ID NO:2 can be generated (Hopp et al., Proc.Natl. Acad. Sci. 78:3824-3828, 1981; Hopp, J. Immun. Meth. 88:1-18, 1986and Triquier et al., Protein Engineering 11:153-169, 1998). The profileis based on a sliding six-residue window. Buried G, S, and T residuesand exposed H, Y, and W residues were ignored. For example, in zsig63,hydrophilic regions include amino acid residues 14 (Phe) to 19 (Arg) ofSEQ ID NO: 2, amino acid residues 16 (Arg) to 21 (Phe) of SEQ ID NO: 2,amino acid residues 24 (Gly) to 29 (Asp) of SEQ ID NO: 2, amino acidresidues 25 (Glu) to 30 (Asp) of SEQ ID NO: 2, and amino acid residues187Glu) to 192 (Glu) of SEQ ID NO: 2.

[0092] Those skilled in the art will recognize that hydrophilicity orhydrophobicity will be taken into account when designing modificationsin the amino acid sequence of a zsig63 polypeptide, so as not to disruptthe overall structural and biological profile. Of particular interestfor replacement are hydrophobic residues selected from the groupconsisting of Val, Leu and Ile or the group consisting of Met, Gly, Ser,Ala, Tyr and Trp. For example, residues tolerant of substitution couldinclude such residues as shown in SEQ ID NO: 2., and described above.

[0093] The identities of essential amino acids can also be inferred fromanalysis of sequence similarity between other known zsig63 proteinfamily members with zsig63. Using methods such as “FASTA” analysisdescribed previously, regions of high similarity are identified within afamily of proteins and used to analyze amino acid sequence for conservedregions. An alternative approach to identifying a variant zsig63polynucleotide on the basis of structure is to determine whether anucleic acid molecule encoding a potential variant zsig63 polynucleotidecan hybridize to a nucleic acid molecule having the nucleotide sequenceof SEQ ID NO:1, as discussed above.

[0094] Other methods of identifying essential amino acids in thepolypeptides of the present invention are procedures known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244:1081 (1989), Bass et al., Proc. NatlAcad. Sci. USA 88:4498 (1991), Coombs and Corey, “Site-DirectedMutagenesis and Protein Engineering,” in Proteins: Analysis and Design,Angeletti (ed.), pages 259-311 (Academic Press, Inc. 1998)). In thelatter technique, single alanine mutations are introduced at everyresidue in the molecule, and the resultant mutant molecules are testedfor biological activity as disclosed below to identify amino acidresidues that are critical to the activity of the molecule. See also,Hilton et al., J. Biol. Chem. 271:4699 (1996).

[0095] The present invention also includes functional fragments ofzsig63 polypeptides and nucleic acid molecules encoding such functionalfragments. A “functional” zsig63 or fragment thereof defined herein ischaracterized by its proliferative or differentiating activity, by itsability to induce or inhibit specialized cell functions, or by itsability to bind specifically to an anti-zsig63antibody or zsig63receptor (either soluble or immobilized).

[0096] Routine deletion analyses of nucleic acid molecules can beperformed to obtain functional fragments of a nucleic acid molecule thatencodes a zsig63 polypeptide. As an illustration, DNA molecules havingthe nucleotide sequence of SEQ ID NO: 1 or fragments thereof, can bedigested with Bal3 1 nuclease to obtain a series of nested deletions.These DNA fragments are then inserted into expression vectors in properreading frame, and the expressed polypeptides are isolated and testedfor zsig63 activity, or for the ability to bind anti-zsig63 antibodiesor zsig63 receptor. One alternative to exonuclease digestion is to useoligonucleotide-directed mutagenesis to introduce deletions or stopcodons to specify production of a desired zsig63 fragment.Alternatively, particular fragments of a zsig63 polynucleotide can besynthesized using the polymerase chain reaction.

[0097] Standard methods for identifying functional domains arewell-known to those of skill in the art. For example, studies on thetruncation at either or both termini of interferons have been summarizedby Horisberger and Di Marco, Pharmac. Ther. 66:507 (1995). Moreover,standard techniques for functional analysis of proteins are describedby, for example, Treuter et al., Molec. Gen. Genet. 240:113 (1993);Content et al., “Expression and preliminary deletion analysis of the 42kDa 2-5A synthetase induced by human interferon,” in BiologicalInterferon Systems, Proceedings of ISIR-TNO Meeting on InterferonSystems, Cantell (ed.), pages 65-72 (Nijhoff 1987); Herschman, “The EGFReceptor,” in Control of Animal Cell Proliferation 1, Boynton et al.,(eds.) pages 169-199 (Academic Press 1985); Coumailleau et al., J. Biol.Chem. 270:29270 (1995); Fukunaga et al., J. Biol. Chem. 270:25291(1995); Yamaguchi et al., Biochem. Pharmacol. 50:1295 (1995); and Meiselet al., Plant Molec. Biol. 30:1 (1996).

[0098] Multiple amino acid substitutions can be made and tested usingknown methods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53-57, 1988) or Bowie and Sauer(Proc. Natl. Acad. Sci. USA 86:2152-2156, 1989). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832-10837, 1991;Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO92/06204) and region-directed mutagenesis (Derbyshire et al., Gene46:145, 1986; Ner et al., DNA 7:127, 1988).

[0099] Variants of the disclosed zsig63 DNA and polypeptide sequencescan be generated through DNA shuffling as disclosed by Stemmer, Nature370:389-91, 1994, Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-51, 1994and WIPO Publication WO 97/20078. Briefly, variant DNAs are generated byin vitro homologous recombination by random fragmentation of a parentDNA followed by reassembly using PCR, resulting in randomly introducedpoint mutations. This technique can be modified by using a family ofparent DNAs, such as allelic variants or DNAs from different species, tointroduce additional variability into the process.

[0100] Selection or screening for the desired activity, followed byadditional iterations of mutagenesis and assay provides for rapid“evolution” of sequences by selecting for desirable mutations whilesimultaneously selecting against detrimental changes.

[0101] Mutagenesis methods as disclosed above can be combined withhigh-throughput, automated screening methods to detect activity ofcloned, mutagenized polypeptides in host cells. Mutagenized DNAmolecules that encode active polypeptides (e.g., anti-microbialactivity) can be recovered from the host cells and rapidly sequencedusing modem equipment. These methods allow the rapid determination ofthe importance of individual amino acid residues in a polypeptide ofinterest, and can be applied to polypeptides of unknown structure.

[0102] In addition, the proteins of the present invention (orpolypeptide fragments thereof) can be joined to other bioactivemolecules, particularly other cytokines, to provide multi-functionalmolecules. For example, one or more helices from zsig63 can be joined toother cytokines to enhance their biological properties or efficiency ofproduction.

[0103] The present invention thus provides a series of novel, hybridmolecules in which a segment comprising one or more of the domains,coil-like structure, or repeats, of zsig63 is fused to anotherpolypeptide. Fusion is preferably done by splicing at the DNA level toallow expression of chimeric molecules in recombinant productionsystems. The resultant molecules are then assayed for such properties asimproved solubility, improved stability, prolonged clearance half-life,improved expression and secretion levels, and pharmacodynamics. Suchhybrid molecules may further comprise additional amino acid residues(e.g. a polypeptide linker) between the component proteins orpolypeptides.

[0104] Using the methods discussed above, one of ordinary skill in theart can identify and/or prepare a variety of polypeptides that aresubstantially similar to residues 16 to 219 of SEQ ID NO:2 or allelicvariants thereof and retain the anti-microbial properties of thewild-type protein. Such polypeptides may include additional amino acidsfrom affinity tags and the like; Such polypeptides may also includeadditional polypeptide segments as generally disclosed above.

[0105] The polypeptides of the present invention, including full-lengthproteins, fragments thereof and fusion proteins, can be produced ingenetically engineered host cells according to conventional techniques.However, host cells must be selected with some care as a result of theanti-microbial activity of the molecules of the present invention. Forexample, any cell culture-based system must be evaluated, because zsig63polypeptides, fragments, fusion proteins, antibodies, agonists orantagonists may kill the host cell as a part of an anti-microbialfunction. Zsig63 polypeptides are of a small enough size to permitpreparation by PCR or other protein chemistry techniques to avoid anypotential host cell toxicity problems. Alternatively, native orengineered precursor proteins, prior to post-translational cleavage toyield the mature zsig63 polypeptide, are inactive, thereby limiting hostcell cytotoxicity prior to lysosomal packaging. See, for example, Lehreret al., Cell 64:229-30, 1991. Thus, precursor proteins to zsig63polypeptides may be produced in microbial cell culture.

[0106] Suitable host cells are those cell types that can be transformedor transfected with exogenous DNA and grown in culture, and includebacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryoticcells, particularly cultured cells of multicellular organisms, arepreferred. Techniques for manipulating cloned DNA molecules andintroducing exogenous DNA into a variety of host cells are disclosed bySambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, andAusubel et al. (eds.), Current Protocols in Molecular Biology, JohnWiley and Sons, Inc., NY, 1987.

[0107] In general, a DNA sequence encoding a zsig63 polypeptide of thepresent invention is operably linked to other genetic elements requiredfor its expression, generally including a transcription promoter andterminator within an expression vector. The vector will also commonlycontain one or more selectable markers and one or more origins ofreplication, although those skilled in the art will recognize thatwithin certain systems selectable markers may be provided on separatevectors, and replication of the exogenous DNA may be provided byintegration into the host cell genome. Selection of promoters,terminators, selectable markers, vectors and other elements is a matterof routine design within the level of ordinary skill in the art. Manysuch elements are described in the literature and are available throughcommercial suppliers.

[0108] To direct a zsig63 polypeptide into the secretory pathway of ahost cell, a secretory signal sequence (also known as a leader sequence,prepro sequence or pre sequence) is provided in the expression vector.The secretory signal sequence may be that of the zsig63 polypeptide,disclosed herein, or may be derived from another secreted protein (e.g.,t-PA) or synthesized de novo. The secretory signal sequence is joined tothe zsig63 polypeptide-encoding DNA sequence in the correct readingframe. Secretory signal sequences are commonly positioned 5′ to the DNAsequence encoding the polypeptide of interest, although certain signalsequences may be positioned elsewhere in the DNA sequence of interest(see, e.g., Welch et al., U.S. Pat. No. 5,037,743; Holland et al., U.S.Pat. No. 5,143,830).

[0109] Alternatively, the secretory signal sequence contained in thepolypeptides of the present invention is used to direct otherpolypeptides into the secretory pathway. The present invention providesfor such fusion polypeptides. A signal fusion polypeptide can be madewherein a secretory signal sequence derived from amino acid residues 1(Met) through 15 (Ala) of SEQ ID NO:2, is operably linked to anotherpolypeptide using methods known in the art and disclosed herein. Thesecretory signal sequence contained in the fusion polypeptides of thepresent invention is preferably fused amino-terminally to an additionalpeptide to direct the additional peptide into the secretory pathway.Such constructs have numerous applications known in the art. Forexample, these novel secretory signal sequence fusion constructs candirect the secretion of an active component of a normally non-secretedprotein. Such fusions may be used In vivo or in vitro to direct peptidesthrough the secretory pathway.

[0110] Cultured mammalian cells are also preferred hosts within thepresent invention. Methods for introducing exogenous DNA into mammalianhost cells include calcium phosphate-mediated transfection (Wigler etal., Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics7:603, 1981: Graham and Van der Eb, Virology 52:456, 1973),electroporation (Neumann et al., EMBO J. 1:841-845, 1982), DEAE-dextranmediated transfection (Ausubel et al., eds., Current Protocols inMolecular Biology, John Wiley and Sons, Inc., NY, 1987),liposome-mediated transfection (Hawley-Nelson et al., Focus 15:73, 1993;Ciccarone et al., Focus 15:80, 1993), and viral vectors (A. Miller andG. Rosman, BioTechniques 7:980-90, 1989; Q. Wang and M. Finer, NatureMed. 2:714-16, 1996). The production of recombinant polypeptides incultured mammalian cells is disclosed, for example, by Levinson et al.,U.S. Pat. No. 4,713,339; Hagen et al., U.S. Pat. No. 4,784,950; Palmiteret al., U.S. Pat. No. 4,579,821; and Ringold, U.S. Pat. No. 4,656,134.Preferred cultured mammalian cells include the COS-1 (ATCC No. CRL1650), COS-7 (ATCC No. CRL 1651), BHK 570 (ATCC No. CRL 10314), 293(ATCC No. CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) andChinese hamster ovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines.Additional suitable cell lines are known in the art and available frompublic depositories such as the American Type Culture Collection,Rockville, Md. In general, strong transcription promoters are preferred,such as promoters from SV-40 or cytomegalovirus. See, e.g., U.S. Pat.No. 4,956,288. Other suitable promoters include those frommetallothionein genes (U.S. Pat. Nos. 4,579,821 and 4,601,978) and theadenovirus major late promoter.

[0111] Drug selection is generally used to select for cultured mammaliancells into which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants”. Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.” Apreferred selectable marker is a gene encoding resistance to theantibiotic neomycin. Selection is carried out in the presence of aneomycin-type drug, such as G-418 or the like. Selection systems mayalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.A preferred amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g., hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used. Alternative markers that introducean altered phenotype, such as green fluorescent protein, or cell surfaceproteins such as CD4, CD8, Class I MHC, placental alkaline phosphatasemay be used to sort transfected cells from untransfected cells by suchmeans as FACS sorting or magnetic bead separation technology.

[0112] Other higher eukaryotic cells can also be used as hosts,including plant cells, insect cells and avian cells. The use ofAgrohacterium rhizogenes as a vector for expressing genes in plant cellshas been reviewed by Sinkar et al., J. Biosci. (Banalore) 11:47-58,1987. Transformation of insect cells and production of foreignpolypeptides therein is disclosed by Guarino et al., U.S. Pat. No.5,162,222 and WIPO publication WO 94/06463. Insect cells can be infectedwith recombinant baculovirus, commonly derived from Autographacaliformica nuclear polyhedrosis virus (AcNPV). DNA encoding the zsig63polypeptide is inserted into the baculoviral genome in place of theAcNPV polyhedrin gene coding sequence by one of two methods. The firstis the traditional method of homologous DNA recombination betweenwild-type AcNPV and a transfer vector containing the zsig63 flanked byAcNPV sequences. Suitable insect cells, e.g. SF9 cells, are infectedwith wild-type AcNPV and transfected with a transfer vector comprising azsig63 polynucleotide operably linked to an AcNPV polyhedrin genepromoter, terminator, and flanking sequences. See, King and Possee, TheBaculovirus Expression System: A Laboratory Guide, London, Chapman &Hall; O'Reilly et al., Baculovirus Expression Vectors: A LaboratoryManual, New York, Oxford University Press., 1994; and, Richardson, C.D., Ed., Baculovirus Expression Protocols. Methods in Molecular Biology,Totowa, N.J., Humana Press, 1995. Natural recombination within an insectcell will result in a recombinant baculovirus which contains zsig63driven by the polyhedrin promoter. Recombinant viral stocks are made bymethods commonly used in the art.

[0113] The second method of making recombinant baculovirus utilizes atransposon-based system described by Luckow (Luckow et al., J. Virol.67:4566-79, 1993). This system is sold in the Bac-to-Bac kit (LifeTechnologies, Rockville, Md.). This system utilizes a transfer vector,pFastBac1™ (Life Technologies) containing a Tn7 transposon to move theDNA encoding the zsig63 polypeptide into a baculovirus genome maintainedin E. coli as a large plasmid called a “bacmid.” The pFastBac1™ transfervector utilizes the AcNPV polyhedrin promoter to drive the expression ofthe gene of interest, in this case zsig63. However, pFastBac1™ can bemodified to a considerable degree. The polyhedrin promoter can beremoved and substituted with the baculovirus basic protein promoter(also known as Pcor, p6.9 or MP promoter) which is expressed earlier inthe baculovirus infection, and has been shown to be advantageous forexpressing secreted proteins. See, Hill-Perkin and Possee, J. Gen.Virol. 71:971-6, 1990; Bonning. et al., J. Gen. Virol. 75:1551-6, 1994;and, Chazenbalk and Rapoport, J. Biol. Chem. 270:1543-9, 1995. In suchtransfer vector constructs, a short or long version of the basic proteinpromoter can be used. Moreover, transfer vectors can be constructedwhich replace the native zsig63 secretory signal sequences withsecretory signal sequences derived from insect proteins. For example, asecretory signal sequence from Ecdysteroid Glucosyltransferase (EGT),honey bee Melittin (Invitrogen, Carlsbad, Calif.), or baculovirus gp67(PharMingen, San Diego, Calif.) can be used in constructs to replace thenative zsig63 secretory signal sequence. In addition, transfer vectorscan include an in-frame fusion with DNA encoding an epitope tag at theC- or N-terminus of the expressed zsig63 polypeptide, for example, aGlu-Glu epitope tag (Grussenmeyer et al., Proc. Natl. Acad. Sci.82:7952-4, 1985). Using a technique known in the art, a transfer vectorcontaining zsig63 is transformed into E. coli, and screened for bacmidswhich contain an interrupted lacZ gene indicative of recombinantbaculovirus. The bacmid DNA containing the recombinant baculovirusgenome is isolated, using common techniques, and used to transfectSpodoptera frugiperda cells, e.g. Sf9 cells. Recombinant virus thatexpresses zsig63 is subsequently produced. Recombinant viral stocks aremade by methods commonly used the art.

[0114] The recombinant virus is used to infect host cells, typically acell line derived from the fall armyworm, Spodoptera frugiperda. See, ingeneral, Glick and Pasternak, Molecular Biotechnology: Principles andApplications of Recombinant DNA, ASM Press, Washington, D.C., 1994.Another suitable cell line is the High FiveO™ cell line (Invitrogen)derived from Trichoplusia ni (U.S. Pat. No. 5,300,435). Commerciallyavailable serum-free media are used to grow and maintain the cells.Suitable media are Sf900 II™ (Life Technologies) or ESF 921™ (ExpressionSystems) for the Sf9 cells; and Ex-cellO405™ (JRH Biosciences, Lenexa,Kans.) or Express FiveO™ (Life Technologies) for the T. ni cells. Thecells are grown up from an inoculation density of approximately 2-5×10⁵cells to a density of 1-2×10⁶ cells at which time a recombinant viralstock is added at a multiplicity of infection (MOI) of 0.1 to 10, moretypically near 3. The recombinant virus-infected cells typically producethe recombinant zsig63 polypeptide at 12-72 hours post-infection andsecrete it with varying efficiency into the medium. The culture isusually harvested 48 hours post-infection. Centrifugation is used toseparate the cells from the medium (supernatant). The supernatantcontaining the zsig63 polypeptide is filtered through micropore filters,usually 0.45 μm pore size. Procedures used are generally described inavailable laboratory manuals (King and Possee, ibid.; O'Reilly et al.,ibid.; Richardson, ibid.). Subsequent purification of the zsig63polypeptide from the supernatant can be achieved using methods describedherein.

[0115] Fungal cells, including yeast cells, can also be used within thepresent invention. Yeast species of particular interest in this regardinclude Saccharomyces cerevisiae, Pichia pastoris, and Pichiamethanolica. Methods for transforming S. cerevisiae cells with exogenousDNA and producing recombinant polypeptides therefrom are disclosed by,for example, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S.Pat. No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S.Pat. No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075.Transformed cells are selected by phenotype determined by the selectablemarker, commonly drug resistance or the ability to grow in the absenceof a particular nutrient (e.g., leucine). A preferred vector system foruse in Saccharomyces cerevisiae is the POT1 vector system disclosed byKawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformedcells to be selected by growth in glucose-containing media. Suitablepromoters and terminators for use in yeast include those from glycolyticenzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman etal., U.S. Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092) andalcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446;5,063,154; 5,139,936 and 4,661,454. Transformation systems for otheryeasts, including Hansenula polymorpha, Schizosaccharomyces pombe,Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichiapastoris, Pichia methanolica, Pichia guillermondii and Candida maltosaare known in the art. See, for example, Gleeson et al., J. Gen.Microbiol. 132:3459-65, 1986 and Cregg, U.S. Pat. No. 4,882,279.Aspergillus cells may be utilized according to the methods of McKnightet al., U.S. Pat. No. 4,935,349. Methods for transforming Acremoniumchrysogenum are disclosed by Sumino et al., U.S. Pat. No. 5,162,228.Methods for transforming Neurospora are disclosed by Lambowitz, U.S.Pat. No. 4,486,533.

[0116] The use of Pichia methanolica as host for the production ofrecombinant proteins is disclosed in WIPO Publications WO 97/17450, WO97/17451, WO 98/02536, and WO 98/02565. DNA molecules for use intransforming P. methanolica will commonly be prepared asdouble-stranded, circular plasmids, which are preferably linearizedprior to transformation. For polypeptide production in P. methanolica,it is preferred that the promoter and terminator in the plasmid be thatof a P. methanolica gene, such as a P. methanolica alcohol utilizationgene (AUG1 or AUG2). Other useful promoters include those of thedihydroxyacetone synthase (DHAS), formate dehydrogenase (FMD), andcatalase (CAT) genes. To facilitate integration of the DNA into the hostchromosome, it is preferred to have the entire expression segment of theplasmid flanked at both ends by host DNA sequences. A preferredselectable marker for use in Pichia methanolica is a P. methanolica ADE2gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC;EC 4.1.1.21), which allows ade2 host cells to grow in the absence ofadenine. For large-scale, industrial processes where it is desirable tominimize the use of methanol, it is preferred to use host cells in whichboth methanol utilization genes (AUG1 and A UG2) are deleted. Forproduction of secreted proteins, host cells deficient in vacuolarprotease genes (PEP4 and PRB1) are preferred. Electroporation is used tofacilitate the introduction of a plasmid containing DNA encoding apolypeptide of interest into P. methanolica cells. It is preferred totransform P. methanolica cells by electroporation using an exponentiallydecaying, pulsed electric field having a field strength of from 2.5 to4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant (t) of from1 to 40 milliseconds, most preferably about 20 milliseconds.

[0117] Prokaryotic host cells, including strains of the bacteriaEscherichia coli, Bacillus and other genera are also useful host cellswithin the present invention.

[0118] Techniques for transforming these hosts and expressing foreignDNA sequences cloned therein are well known in the art (see, e.g.,Sambrook et al., ibid.). When expressing a zsig63 polypeptide inbacteria such as E. coli, the polypeptide may be retained in thecytoplasm, typically as insoluble granules, or may be directed to theperiplasmic space by a bacterial secretion sequence. In the former case,the cells are lysed, and the granules are recovered and denatured using,for example, guanidine isothiocyanate or urea. The denatured polypeptidecan then be refolded and dimerized by diluting the denaturant, such asby dialysis against a solution of urea and a combination of reduced andoxidized glutathione, followed by dialysis against a buffered salinesolution. In the latter case, the polypeptide can be recovered from theperiplasmic space in a soluble and functional form by disrupting thecells (by, for example, sonication or osmotic shock) to release thecontents of the periplasmic space and recovering the protein, therebyobviating the need for denaturation and refolding.

[0119] Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media may alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell. P. methanolicacells are cultured in a medium comprising adequate sources of carbon,nitrogen and trace nutrients at a temperature of about 25° C. to 35° C.Liquid cultures are provided with sufficient aeration by conventionalmeans, such as shaking of small flasks or sparging of fermentors. Apreferred culture medium for P. methanolica is YEPD (2% D-glucose, 2%Bacto™ Peptone (Difco Laboratories, Detroit, Mich.), 1% Bacto™ yeastextract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine).

[0120] Expressed recombinant zsig63 polypeptides (or chimeric zsig63polypeptides) can be purified using fractionation and/or conventionalpurification methods and media. Ammonium sulfate precipitation and acidor chaotrope extraction may be used for fractionation of samples.Exemplary purification steps may include hydroxyapatite, size exclusion,FPLC and reverse-phase high performance liquid chromatography. Suitableanion exchange media include derivatized dextrans, agarose, cellulose,polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Qderivatives are preferred, with DEAE Fast-Flow Sepharose (Pharmacia,Piscataway, N.J.) being particularly preferred. Exemplarychromatographic media include those media derivatized with phenyl,butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia),Toyopearl butyl 650 (Toso Haas, Montgomeryville, Pa.), Octyl-Sepharose(Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG71 (Toso Haas) and the like. Suitable solid supports include glassbeads, silica-based resins, cellulosic resins, agarose beads,cross-linked agarose beads, polystyrene beads, cross-linkedpolyacrylamide resins and the like that are insoluble under theconditions in which they are to be used.

[0121] These supports may be modified with reactive groups that allowattachment of proteins by amino groups, carboxyl groups, sulfhydrylgroups, hydroxyl groups and/or carbohydrate moieties. Examples ofcoupling chemistries include cyanogen bromide activation,N-hydroxysuccinimide activation, epoxide activation, sulfhydrylactivation, hydrazide activation, and carboxyl and amino derivatives forcarbodiimide coupling chemistries. These and other solid media are wellknown and widely used in the art, and are available from commercialsuppliers. Methods for binding receptor polypeptides to support mediaare well known in the art. Selection of a particular method is a matterof routine design and is determined in part by the properties of thechosen support. See, for example, Affinity Chromatography: Principles &Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988.

[0122] The polypeptides of the present invention can be isolated byexploitation of their structural and biological properties. For example,immobilized metal ion adsorption (IMAC) chromatography can be used topurify histidine-rich proteins or proteins having a His-affinity tag.Briefly, a gel is first charged with divalent metal ions to form achelate (Sulkowski, Trends in Biochem. 3:1-7, 1985). Histidine-richproteins will be adsorbed to this matrix with differing affinities,depending upon the metal ion used, and will be eluted by competitiveelution, lowering the pH, or use of strong chelating agents. Othermethods of purification include purification of glycosylated proteins bylectin affinity chromatography and ion exchange chromatography (Methodsin Enzymol., Vol. 182, “Guide to Protein Purification”, M. Deutscher,(ed.), Acad. Press, San Diego, 1990, pp.529-39). Within additionalembodiments of the invention, a fusion of the polypeptide of interestand an affinity tag (e.g., maltose-binding protein, an immunoglobulindomain) may be constructed to facilitate purification.

[0123] Protein refolding (and optionally reoxidation) procedures may beadvantageously used. It is preferred to purify the protein to >80%purity, more preferably to >90% purity, even more preferably >95%, andparticularly preferred is a pharmaceutically pure state, that is greaterthan 99.9% pure with respect to contaminating macromolecules,particularly other proteins and nucleic acids, and free of infectiousand pyrogenic agents. Preferably, a purified protein is substantiallyfree of other proteins, particularly other proteins of animal origin.

[0124] zsig63 polypeptides or fragments thereof may also be preparedthrough chemical synthesis. zsig63 polypeptides may be monomers ormultimers; glycosylated or non-glycosylated; pegylated or non-pegylated;amidated or non-amidated; sulfated or non-sulfated; and may or may notinclude an initial methionine amino acid residue.

[0125] Polypeptides of the present invention can also be synthesized byexclusive solid phase synthesis, partial solid phase methods, fragmentcondensation or classical solution synthesis. Methods for synthesizingpolypeptides are well known in the art. See, for example, Merrifield, J.Am. Chem. Soc. 85:2149, 1963; Kaiser et al., Anal. Biochem. 34:595,1970. After the entire synthesis of the desired peptide on a solidsupport, the peptide-resin is washed with a reagent which cleaves thepolypeptide from the resin and removes most of the side-chain protectinggroups. Such methods are well established in the art.

[0126] Molecules of the present invention can be used to identify andisolate receptors that bind zsig63 polypeptide. For example, proteinsand peptides of the present invention can be immobilized on a column andmembrane preparations run over the column (Immobilized Affinity LigandTechniques, Hermanson et al., eds., Academic Press, San Diego, Calif.,1992, pp.195-202). Proteins and peptides can also be radiolabeled(Methods in Enzymol., vol. 182, “Guide to Protein Purification”, M.Deutscher, ed., Acad. Press, San Diego, 1990, 721-37) or photoaffinitylabeled (Brunner et al., Ann. Rev. Biochem. 62:483-514, 1993 and Fedanet al., Biochem. Pharmacol. 33:1167-80, 1984) and specific cell-surfaceproteins can be identified.

[0127] An assay system that uses a ligand-binding receptor (or anantibody, one member of a complement/anti-complement pair) or a bindingfragment thereof, and a commercially available biosensor instrument(BIAcore™, Pharmacia Biosensor, Piscataway, N.J.) may be advantageouslyemployed. Such receptor, antibody, member of acomplement/anti-complement pair or fragment is immobilized onto thesurface of a receptor chip. Use of this instrument is disclosed byKarlsson, J. Immunol. Methods 145:229-40,1991 and Cunningham and Wells,J. Mol. Biol. 234:554-63, 1993. A receptor, antibody, member or fragmentis covalently attached, using amine or sulfhydryl chemistry, to dextranfibers that are attached to gold film within the flow cell. A testsample is passed through the cell. If a ligand, epitope, or oppositemember of the complement/anti-complement pair is present in the sample,it will bind to the immobilized receptor, antibody or member,respectively, causing a change in the refractive index of the medium,which is detected as a change in surface plasmon resonance of the goldfilm. This system allows the determination of on- and off-rates, fromwhich binding affinity can be calculated, and assessment ofstoichiometry of binding.

[0128] Ligand-binding receptor polypeptides can also be used withinother assay systems known in the art. Such systems include Scatchardanalysis for determination of binding affinity (see Scatchard, Ann. NYAcad. Sci. 51:660-72, 1949) and calorimetric assays (Cunningham et al.,Science 253:545-48, 1991; Cunningham et al., Science 245:821-25, 1991).

[0129] In another embodiment, polypeptide-toxin fusion proteins orantibody-toxin fusion proteins can be used for targeted cell or tissueinhibition or ablation (for instance, to treat cancer cells or tissues).Alternatively, if the polypeptide has multiple functional domains (i.e.,an activation domain or a receptor binding domain, plus a targetingdomain), a fusion protein including only the targeting domain may besuitable for directing a detectable molecule, a cytotoxic molecule or acomplementary molecule to a cell or tissue type of interest. Ininstances where the domain only fusion protein includes a complementarymolecule, the anti-complementary molecule can be conjugated to adetectable or cytotoxic molecule. Such domain-complementary moleculefusion proteins thus represent a generic targeting vehicle forcell/tissue-specific delivery of genericanti-complementary-detectable/cytotoxic molecule conjugates.

[0130] In another embodiment, zsig63-cytokine fusion proteins orantibody-cytokine fusion proteins can be used for enhancing in vivokilling of target tissues (for example, blood and bone marrow cancers),if the zsig63 polypeptide or anti-zsig63 antibody targets thehyperproliferative blood or bone marrow cell (See, generally, Homick etal., Blood 89:4437-47, 1997). Homick et al. described fusion proteinsthat target a cytokine to a desired site of action, thereby providing anelevated local concentration of cytokine. Suitable zsig63 polypeptidesor anti-zsig63 antibodies can target an undesirable cell or tissue(i.e., a tumor or a leukemia), and the fused cytokine can mediateimproved target cell lysis by effector cells. Suitable cytokines forthis purpose include interleukin 2 and granulocyte-macrophagecolony-stimulating factor (GM-CSF), for instance.

[0131] In yet another embodiment, if the zsig63 polypeptide oranti-zsig63 antibody targets vascular cells or tissues, such polypeptideor antibody may be conjugated with a radionuclide, and particularly witha beta-emitting radionuclide, to reduce restenosis. Such therapeuticapproach poses less danger to clinicians who administer the radioactivetherapy. For instance, iridium-192 impregnated ribbons placed intostented vessels of patients until the required radiation dose wasdelivered showed decreased tissue growth in the vessel and greaterluminal diameter than the control group, which received placebo ribbons.Further, revascularisation and stent thrombosis were significantly lowerin the treatment group. Similar results are predicted with targeting ofa bioactive conjugate containing a radionuclide, as described herein.

[0132] The bioactive polypeptide or antibody conjugates described hereincan be delivered intravenously, intraarterially or intraductally, or maybe introduced locally at the intended site of action.

[0133] Another aspect of the present invention includes zsig63polypeptide fragments. Preferred fragments include the leader sequence,ranging from amino acid 1 (Met) to amino acid 15 (Ala) of SEQ ID NO:2.Such leader sequences may be used to direct the secretion of otherpolypeptides. Such fragments of the present invention may be used asfollows: the alternative secretion leader fragments are formed as fusionproteins with alternative proteins selected for secretion; plasmidsbearing regulatory regions capable of directing the expression of thefusion protein are introduced into test cells; and secretion of theprotein is monitored.

[0134] Moreover, using methods described in the art, polypeptidefusions, or hybrid zsig63 proteins, are constructed using regions ordomains of zsig63 in combination with those of paralogs, orthologs, orheterologous proteins (Sambrook et al., ibid., Altschul et al., ibid.,Picard. D., Cur. Opin. Biology, 5:511-515, 1994, and referencestherein). These methods allow the determination of the biologicalimportance of larger domains or regions in a polypeptide of interest.Such hybrids may alter reaction kinetics, binding, constrict or expandthe substrate specificity, or alter tissue and cellular localization ofa polypeptide, and can be applied to polypeptides of unknown structure.

[0135] Fusion polypeptides can be prepared by methods known to thoseskilled in the art by preparing each component of the fusion protein andchemically conjugating them. Alternatively, a polynucleotide encodingone or more components of the fusion protein in the proper reading framecan be generated using known techniques and expressed by the methodsdescribed herein. For example, part or all of a domain(s) conferring abiological function may be swapped between zsig63 of the presentinvention with the functionally equivalent domain(s) from another familymember. Such domains include, but are not limited to the secretorysignal sequence, and domains 1 through 3, and the coil-like structure,described herein. Such fusion proteins would be expected to have abiological functional profile that is the same or similar topolypeptides of the present invention or other known family proteins orto a heterologous protein, depending on the fusion constructed.Moreover, such fusion proteins may exhibit other properties as disclosedherein.

[0136] Standard molecular biological and cloning techniques can be usedto swap the equivalent domains between the zsig63 polypeptide and thosepolypeptides to which they are fused. Generally, a DNA segment thatencodes a domain of interest, e.g., a zsig63 domain 1, 2, or 3, or amotif described herein, is operably linked in frame to at least oneother DNA segment encoding an additional polypeptide and inserted intoan appropriate expression vector, as described herein. Generally DNAconstructs are made such that the several DNA segments that encode thecorresponding regions of a polypeptide are operably linked in frame tomake a single construct that encodes the entire fusion protein, or afunctional portion thereof. For example, a DNA construct would encodefrom N-terminus to C-terminus a fusion protein comprising a signalpolypeptide followed by a mature polypeptide; or a DNA construct wouldencode from N-terminus to C-terminus a fusion protein comprising asignal polypeptide followed by domain-1, followed by domain-2, followedby domain-3, or as interchanged with equivalent regions from anotherprotein. Such fusion proteins can be expressed, isolated, and assayedfor activity as described herein. Polypeptide linkers are preferablyemployed if necessary to provide separation of component polypeptides ofthe fusion or to allow for flexibility of the fusion protein, therebypreserving the anti-microbial activity of each defensin component of thefusion protein. Those of ordinary skill in the art are capable ofdesigning such linkers.

[0137] Within another aspect of the present invention there is provideda pharmaceutical composition comprising purified zsig63 polypeptide incombination with a pharmaceutically acceptable vehicle. Suchpharmaceutical compositions are used in the treatment of conditionsassociated with pathological microbes, including bacterial, fungal andviral infections. Antibacterial applications of zsig63 polypeptideinclude situations where the pathogen has become resistant to standardtreatments. For example, hospital sepsis is an increasing problem, sinceStaphylococcus and other bacterial and microbial strains have becomeresistant to commonly used antibiotics.

[0138] In general, anti-microbial activity of zsig63 polypeptides,fragments, fusions, antibodies, agonists and antagonists can beevaluated by techniques that are known in the art. More specifically,anti-microbial activity can be assayed by evaluating the sensitivity ofmicrobial cell cultures to test agents and by evaluating the protectiveeffect of test agents on infected mice. See, for example, Musiek et al.,Antimicrob. Agents Chemothr. 3:40, 1973. Antiviral activity can also beassessed by protection of mammalian cell cultures. Known techniques forevaluating anti-microbial activity include, for example, Barsum et al.,Eur. Respir. J. 8(5): 709-14, 1995; Sandovsky-Losica et al., J. Med.Vet. Mycol (England) 28(4):279-87, 1990; Mehentee et al., J. Gen.Microbiol (England) 135 (Pt. 8): 2181-8, 1989; Segal and Savage, Journalof Medical and Veterinary Mycology 24:477-479, 1986 and the like. Knownassays specific for anti-viral activity include, for example, thosedescribed by Daher et al., J. Virol. 60(3): 1068-74, 1986.

[0139] In addition, contract laboratories offer services in evaluatinganti-microbial properties. For example, Panlabs, Inc. of Bothell,Washington offer in vitro or in vivo testing for bacteria, gram negative(Enterobacter cloacae, Escherichia coli, Klebsiella pneumonia, Proteusvulgaris, Pseudomonas aeruginosa, Salmonella typhimurium and Serratiamarcescens), gram positive (Bacillus subtilis, Brevebacteriumammoniagenes, Corynebacterium minutissimum, Micrococcus luteus,Mycobacterium ranae, Staphylococcus strains and Streptococcus strains)and anaerobic organisms (Actinomyces viscosus, Bacteroides fragilis,Clostridium sporogenes, Corynebacterium acnes, Helicobacter pylori andPorphyromonas gingivalis), as well as for protozoa (Trichomonas foetus)and fungi (e.g., Candida albicans, Epidermophyton floccosum, Exophialajeanselmei, Microsporum strains, Trichophyton strains and the like).Also, Molecular Probes of Oregon has commercially available fluorescencetechnology for use in bacteriology.

[0140] If desired, zsig63 polypeptide, fragment, fusion protein,agonist, antagonist or antibody performance in this regard can becompared to proteins known to be functional in this regard, such asproline-rich proteins, lysozyme, histatins, lactoperoxidase or the like.In addition, zsig63 polypeptide, fragment, fusion protein, antibody,agonist or antagonist may be evaluated in combination with one or moreanti-microbial agents to identify synergistic effects.

[0141] High expression of zsig63 in salivary gland suggests thatanti-microbial polypeptides of the present invention can be used fortreatment of dental carries (tooth decay), periodontal disease, thrush,and gastrointestinal disease. Other applications can be used in urinarytract infections, vaginal infections, prevention of infection in skinand other epithelial wounds. As such, the polypeptides of the presentinvention can help establish normal microflora and protect againstpathogenic colonization and invasion.

[0142] Moreover, microorganisms have specific infective stages wherethey decorate their surface with proline coil containing proteins. Onetheory is that they are decoying or evading the host immune system byexpressing host-like, but inactive, proteins on their surfaces. Thezsig63 polypeptide of the present invention can be one of these hostpolypeptides that the microorganism is mimicking. Moreover, there may bea correlation with Zinc and resistance to microbial infection. If zsig63binds zinc (or some other cation) there may be a delivery function, orimmune activation function with zinc as a co-factor for regulation ofspecific cell types, or defensive enzymes. Such immune activation byzsig63 polypeptides can be assessed by assays that are well known in theart.

[0143] The pharmaceutical compositions of the present invention may alsobe used when pro-inflammatory activity is desired. Applications for suchpro-inflammatory activity include the treatment of chronic tissuedamage, particularly in areas having a limited or damaged vascularsystem, e.g., damage in extremities associated with diabetes. Incontrast, antagonists to zsig63 polypeptides may be useful asanti-inflammatory agents.

[0144] Zsig63 polypeptide pharmaceutical compositions of the presentinvention may also be used in the treatment of conditions wherestimulation of immune responsiveness is desired. Such conditions includethe treatment of patients having incompetent immune systems, such asAIDS patients or individuals that have undergone chemotherapy, radiationtreatment or the like.

[0145] Because zsig63 polypeptide was found in a salivary gland libraryand cystic fibrosis is characterized by frequent microbial infection,pharmaceutical compositions containing zsig63 polypeptide are alsocontemplated for use in the treatment of lung infections associated withcystic fibrosis. Also contemplated by the present invention areengineered zsig63 polypeptides that are characterized by decreasedsensitivity to salt concentration. Decreased sensitivity to high saltconcentration will preserve anti-microbial activity of engineered zsig63polypeptides in high salt environments, such as in the lung airways ofpatients suffering from cystic fibrosis. In this manner, pharmaceuticalcompositions containing engineered zsig63 polypeptides that areformulated for delivery to the lungs can be used to treat lunginfections associated with cystic fibrosis.

[0146] Another aspect of the present invention involves the detection ofzsig63 polypeptides in cell culture or in a serum sample or tissuebiopsy of a patient undergoing evaluation for SPG, Chediak-Higashisyndrome, or other conditions characterized by an alteration in defensinconcentration. Zsig63 polypeptides can be detected using immunoassaytechniques and antibodies capable of recognizing a zsig63 polypeptideepitope, as described herein. More specifically, the present inventioncontemplates methods for detecting zsig63 polypeptide comprising:

[0147] exposing a solution or sample or cell culture lysate orsupernatant, possibly containing zsig63 polypeptide, to an antibodyattached to a solid support, wherein said antibody binds to a firstepitope of a zsig63 polypeptide;

[0148] washing said immobilized antibody-polypeptide to remove unboundcontaminants;

[0149] exposing the immobilized antibody-polypeptide to a secondantibody directed to a second epitope of a zsig63 polypeptide, whereinthe second antibody is associated with a detectable label; and

[0150] detecting the detectable label. Zsig63 polypeptide concentrationdiffering from that of controls may be indicative of SPG,Chediak-Higashi syndrome or other conditions characterized by analteration in defensin concentration. In addition, expression of zsig63may be monitored in cystic fibrosis patients as a predictor of the onsetof infectious crises. Also, high defensin levels have been associatedwith cytotoxic effects in lung, indicating that other hostanti-microbial polypeptides, such as zsig63 polypeptide levels can beused as indicators for disease onset and cytotoxicity, and used todirect treatment for averting or addressing such cytotoxicity. Forexample, antibodies directed to zsig63 polypeptide can be administeredto inactivate the same in a treatment modality.

[0151] Within additional aspects of the invention there are providedantibodies or synthesized binding proteins(e.g., those generated byphage display, E. coli Fab, and the like) that specifically bind to thezsig63 polypeptides described above. Such antibodies are useful for,among other uses as described herein, preparation of anti-idiotypicantibodies. Synthesized binding proteins may be produced by phagedisplay using commercially available kits, such as the Ph.D.™ PhageDisplay Peptide Library Kits available from New England Biolabs, Inc.(Beverly, Mass.). Phage display techniques are described, for example,in U.S. Pat. Nos. 5,223,409, 5,403,484 and 5,571,698.

[0152] An additional aspect of the present invention provides methodsfor identifying agonists or antagonists of the zsig63 polypeptidesdisclosed above, which agonists or antagonists may have valuableproperties as discussed further herein. Within one embodiment, there isprovided a method of identifying zsig63 polypeptide agonists, comprisingproviding cells responsive thereto, culturing the cells in the presenceof a test compound and comparing the cellular response with the cellcultured in the presence of the zsig63 polypeptide, and selecting thetest compounds for which the cellular response is of the same type.

[0153] Within another embodiment, there is provided a method ofidentifying antagonists of zsig63 polypeptide, comprising providingcells responsive to a zsig63 polypeptide, culturing a first portion ofthe cells in the presence of zsig63 polypeptide, culturing a secondportion of the cells in the presence of the zsig63 polypeptide and atest compound, and detecting a decrease in a cellular response of thesecond portion of the cells as compared to the first portion of thecells.

[0154] A further aspect of the invention provides a method of studyingchemoattraction of monocytes in cell culture, comprising incubatingmonocytes in a culture medium comprising a zsig63 polypeptide, fragment,fusion protein, antibody, agonist or antagonist to study or evaluatemonocyte chemoattraction. Such evaluation may be conducted using methodsknown in the art, such as those described by Territo et al. referencedabove.

[0155] Melanocortin receptors are G-coupled protein receptors whichactivate adenylate cyclase and cause calcium flux. The agouti protein(which contains a 36 amino acid domain that is toxin-like) is thought toinhibit the binding of MSH-alpha to MC1 and MC4. In addition, the agoutiprotein is thought to be an antagonist of calcium channels, and certaintoxins are believed to modulate ion flux. Experimental evidence has beengenerated, suggesting that defensins are capable of blocking calciumchannels. Similarly, zsig63 polypeptides could have such properties.

[0156] A further aspect of the invention provides a method of studyingactivity of the melanocortin family of receptors in cell culture,comprising incubating cells that endogenously bear such receptors (e.g.,ACTH receptors or the like) or cells that have been engineered to bearsuch receptors in a culture medium comprising a ligand or putativeligand and zsig63 polypeptide, fragment, fusion protein, antibody,agonist or antagonist to study or evaluate ligand or putative ligandbinding and/or ion flux regulation or modulation. Such evaluation may beconducted using methods known in the art, such as those described by Zhuet al. referenced above.

[0157] A further aspect of the invention provides a method of studyingion flux in cell culture, comprising incubating cells that are capableof ion flux, such as calcium flux, sodium flux, potassium flux or thelike, in a culture medium comprising zsig63 polypeptide, fragment,fusion protein, antibody, agonist or antagonist to study or evaluate ionflux regulation or modulation.

[0158] A further aspect of the invention provides a method of studyingcytocidal activity against mammalian cells, such as tumor cells, in cellculture, comprising incubating such cells in a culture medium comprisinga zsig63 polypeptide, fragment, fusion protein, antibody, agonist orantagonist at high test agent and low cell concentration to study orevaluate cytocidal activity. Such evaluation may be conducted usingmethods known in the art, such as those described by Lichtenstein etal., Blood 68:1407-10, 1986 and Sheu et al., Antimicrob. AgentsChemother. 28:626-9, 1993.

[0159] Another aspect of the present invention involves the use ofzsig63 polypeptides, fragments, fusion proteins or agonists as cellculture reagents in in vitro studies of exogenous microorganisminfection, such as bacterial, viral or fungal infection. Such moietiesmay also be used in in vivo animal models of infection.

[0160] An additional aspect of the present invention is to studyepithelial cell defensin induction in cell culture. In this aspect ofthe present invention, epithelial cells are cultured and exposed topathogenic stimuli. Induction of zsig63 polypeptide production by theepithelial cells is then measured.

[0161] A high level of expression of zsig63 polypeptide was observed byNorthern blot in the trachea and by dot blot in the salivary gland andtrachea. Consequently, another aspect of the present invention involvesthe detection of zsig63 polypeptides in the serum or tissue biopsy of apatient undergoing evaluation for salivary gland function ordysfunction. Such zsig63 polypeptides can be detected using immunoassaytechniques and antibodies capable of recognizing zsig63 polypeptideepitopes.

[0162] More specifically, the present invention contemplates methods fordetecting zsig63 polypeptide comprising:

[0163] exposing a solution possibly containing zsig63 polypeptide to anantibody attached to a solid support, wherein said antibody binds to afirst epitope of a zsig63 polypeptide;

[0164] washing said immobilized antibody-polypeptide to remove unboundcontaminants;

[0165] exposing the immobilized antibody-polypeptide to a secondantibody directed to a second epitope of a zsig63 polypeptide, whereinthe second antibody is associated with a detectable label; and

[0166] detecting the detectable label. Changes in serum or biopsy zsig63polypeptide concentration (relative to normal serum or tissueconcentration) may be indicative of dysfunction of the salivary gland.

[0167] Salivary gland dysfunction includes digestive dysfunction, woundhealing dysfunction, inadequate saliva production or composition,mucosal integrity breakdown, and failure of or diminished anti-microbialfunction. Detection of zsig63 polypeptide at relatively high levels inthe trachea may indicate that such polypeptides may serve as a marker oflung dysfunction. Moreover, zsig63 expression is detected in lung.Examples of conditions associated with salivary gland or lungdysfunction include salivary gland carcinoma, sarcoidosis, pneumocysticcarinii (particularly as associated with AIDS patients), emphysema,chronic bronchitis, cystic fibrosis, ARDS, SIDS or the like. Inaddition, zsig63 polypeptides are expressed in the prostate at a levelsimilar to trachea, as well as in the salivary gland. The prostate glandis androgen regulated and shares other properties with salivary glands.Consequently, dysfunction thereof, such as prostate adenocarcinoma orthe like, may also be detected using zsig63 polypeptides or zsig63antibodies.

[0168] Also, the salivary glands synthesize and secrete a number ofproteins having diverse biological functions. Such proteins facilitatelubrication of the oral cavity (e.g., mucins and proline-rich proteins),remineralization (e.g., statherin and ionic proline-rich proteins) anddigestion (e.g., amylase, lipase and proteases) and provideanti-microbial (e.g., proline-rich proteins, lysozyme, histatins andlactoperoxidase) and mucosal integrity maintenance (e.g., mucins)capabilities. In addition, saliva is a rich source of growth factorssynthesized by the salivary glands. For example, saliva is known tocontain epidermal growth factor (EGF), nerve growth factor (NGF),transforming growth factor-alpha (TGF-α), transforming growthfactor-beta (TGF-β), insulin, insulin-like growth factors I and II(IGF-I and IGF-II) and fibroblast growth factor (FGF). See, for example,Zelles et al., J. Dental. Res. 74(12): 1826-32, 1995. Synthesis ofgrowth factors by the salivary gland is believed to beandrogen-dependent and to be necessary for the health of the oral cavityand gastrointestinal tract.

[0169] Thus, zsig63 polypeptides, agonists or antagonists thereof may betherapeutically useful for aiding digestion. To verify the presence ofthis capability in zsig63 polypeptides, agonists or antagonists of thepresent invention, such zsig63 polypeptides, agonists or antagonists areevaluated with respect to their ability to break down starch accordingto procedures known in the art. If desired, zsig63 polypeptideperformance in this regard can be compared to digestive enzymes, such asamylase, lipase, proteases and the like. In addition, zsig63polypeptides or agonists or antagonists thereof may be evaluated incombination with one or more digestive enzymes to identify synergisticeffects.

[0170] The activity of molecules of the present invention can bemeasured using a variety of assays that measure stimulation ofgastrointestinal cell contractility, modulation of nutrient uptakeand/or secretion of digestive enzymes. Of particular interest arechanges in contractility of smooth muscle cells. For example, thecontractile response of segments of mammalian duodenum or othergastrointestinal smooth muscles tissue (Depoortere et al., J.Gastrointestinal Motility 1:150-159, 1989). An exemplary in vivo assayuses an ultrasonic micrometer to measure the dimensional changesradially between commissures and longitudinally to the plane of thevalve base (Hansen et al., Society of Thoracic Surgeons 60:S384-390,1995).

[0171] An in vivo approach for assaying proteins of the presentinvention involves viral delivery systems. Exemplary viruses for thispurpose include adenovirus, herpesvirus, retroviruses, vaccinia virus,and adeno-associated virus (AAV). Adenovirus, a double-stranded DNAvirus, is currently the best studied gene transfer vector for deliveryof heterologous nucleic acid (for review, see T. C. Becker et al., Meth.Cell Biol. 43:161-89, 1994; and J. T. Douglas and D. T. Curiel, Science& Medicine 4:44-53, 1997). The adenovirus system offers severaladvantages: (i) adenovirus can accommodate relatively large DNA inserts;(ii) can be grown to high-titer; (iii) infect a broad range of mammaliancell types; and (iv) can be used with many different promoters includingubiquitous, tissue specific, and regulatable promoters. Also, becauseadenoviruses are stable in the bloodstream, they can be administered byintravenous injection.

[0172] Using adenovirus vectors where portions of the adenovirus genomeare deleted, inserts are incorporated into the viral DNA by directligation or by homologous recombination with a co-transfected plasmid.In an exemplary system, the essential E1 gene has been deleted from theviral vector, and the virus will not replicate unless the E1 gene isprovided by the host cell (the human 293 cell line is exemplary). Whenintravenously administered to intact animals, adenovirus primarilytargets the liver. If the adenoviral delivery system has an E 1 genedeletion, the virus cannot replicate in the host cells. However, thehost's tissue (e.g., liver) will express and process (and, if asecretory signal sequence is present, secrete) the heterologous protein.Secreted proteins will enter the circulation in the highly vascularizedliver, and effects on the infected animal can be determined.

[0173] Moreover, adenoviral vectors containing various deletions ofviral genes can be used in an attempt to reduce or eliminate immuneresponses to the vector. Such adenoviruses are El deleted, and inaddition contain deletions of E2A or E4 (Lusky, M. et al., J. Virol.72:2022-2032, 1998; Raper, S. E. et al., Human Gene Therapy 9:671-679,1998). In addition, deletion of E2b is reported to reduce immuneresponses (Amalfitano, A. et al., J. Virol. 72:926-933, 1998). Moreover,by deleting the entire adenovirus genome, very large inserts ofheterologous DNA can be accommodated. Generation of so called “gutless”adenoviruses where all viral genes are deleted are particularlyadvantageous for insertion of large inserts of heterologous DNA. Forreview, see Yeh, P. and Perricaudet, M., FASEB J. 11:615-623, 1997.

[0174] The adenovirus system can also be used for protein production invitro. By culturing adenovirus-infected non-293 cells under conditionswhere the cells are not rapidly dividing, the cells can produce proteinsfor extended periods of time. For instance, BHK cells are grown toconfluence in cell factories, then exposed to the adenoviral vectorencoding the secreted protein of interest. The cells are then grownunder serum-free conditions, which allows infected cells to survive forseveral weeks without significant cell division. Alternatively,adenovirus vector infected 293 cells can be grown as adherent cells orin suspension culture at relatively high cell density to producesignificant amounts of protein (See Garnier et al., Cytotechnol.15:145-55, 1994). With either protocol, an expressed, secretedheterologous protein can be repeatedly isolated from the cell culturesupernatant, lysate, or membrane fractions depending on the dispositionof the expressed protein in the cell. Within the infected 293 cellproduction protocol, non-secreted proteins may also be effectivelyobtained.

[0175] Compounds identified as zsig63 agonists are useful in vitro andin vivo. For example, zsig63 and agonist compounds are useful ascomponents of defined cell culture media, and may be used alone or incombination with other cytokines and hormones to replace serum that iscommonly used in cell culture. Thus, zsig63 polypeptides and zsig63agonist polypeptides are useful as a research reagent, such as for theexpansion of cultured cells. As such, zsig63 polypeptides are added totissue culture media for these cell types.

[0176] As a ligand, the activity of zsig63 polypeptide can be measuredby a silicon-based biosensor microphysiometer which measures theextracellular acidification rate or proton excretion associated withreceptor binding and subsequent physiologic cellular responses. Anexemplary device is the Cytosensor™ Microphysiometer manufactured byMolecular Devices, Sunnyvale, Calif. A variety of cellular responses,such as cell proliferation, ion transport, energy production,inflammatory response, regulatory and receptor activation, and the like,can be measured by this method. See, for example, McConnell, H. M. etal., Science 257:1906-1912, 1992; Pitchford, S. et al., Meth. Enzymol.228:84-108, 1997; Arimilli, S. et al., J. Immunol. Meth. 212:49-59,1998; Van Liefde, I. et al., Eur. J.

[0177] Pharmacol. 346:87-95, 1998. The microphysiometer can be used forassaying adherent or non-adherent eukaryotic or prokaryotic cells. Bymeasuring extracellular acidification changes in cell media over time,the microphysiometer directly measures cellular responses to variousstimuli, including zsig63 polypeptide, its agonists, or antagonists.Preferably, the microphysiometer is used to measure responses of azsig63-responsive eukaryotic cell, compared to a control eukaryotic cellthat does not respond to zsig63 polypeptide. Zsig63-responsiveeukaryotic cells comprise cells into which a receptor for zsig63 hasbeen transfected creating a cell that is responsive to zsig63; or cellsnaturally responsive to zsig63 such as cells derived from, for example,pancreas, intestinal, prostate or tracheal tissue. Differences, measuredby a change, for example, an increase or diminution in extracellularacidification, in the response of cells exposed to zsig63 polypeptide,relative to a control not exposed to zsig63, are a direct measurement ofzsig63-modulated cellular responses. Moreover, such zsig63-modulatedresponses can be assayed under a variety of stimuli. Using themicrophysiometer, there is provided a method of identifying agonists ofzsig63 polypeptide, comprising providing cells responsive to a zsig63polypeptide, culturing a first portion of the cells in the absence of atest compound, culturing a second portion of the cells in the presenceof a test compound, and detecting a change, for example, an increase ordiminution, in a cellular response of the second portion of the cells ascompared to the first portion of the cells. The change in cellularresponse is shown as a measurable change extracellular acidificationrate. Moreover, culturing a third portion of the cells in the presenceof zsig63 polypeptide and the absence of a test compound can be used asa positive control for the zsig63-responsive cells, and as a control tocompare the agonist activity of a test compound with that of the zsig63polypeptide. Moreover, using the microphysiometer, there is provided amethod of identifying antagonists of zsig63 polypeptide, comprisingproviding cells responsive to a zsig63 polypeptide, culturing a firstportion of the cells in the presence of zsig63 and the absence of a testcompound, culturing a second portion of the cells in the presence ofzsig63 and the presence of a test compound, and detecting a change, forexample, an increase or a diminution in a cellular response of thesecond portion of the cells as compared to the first portion of thecells. The change in cellular response is shown as a measurable changeextracellular acidification rate. Antagonists and agonists, for zsig63polypeptide, can be rapidly identified using this method.

[0178] Moreover, zsig63 can be used to identify cells, tissues, or celllines which respond to a zsig63-stimulated pathway. Themicrophysiometer, described above, can be used to rapidly identifyligand-responsive cells, such as cells responsive to zsig63 of thepresent invention. Cells can be cultured in the presence or absence ofzsig63 polypeptide. Those cells which elicit a measurable change inextracellular acidification in the presence of zsig63 are responsive tozsig63. Such cells, can be used to identify antagonists and agonists ofzsig63 polypeptide as described above.

[0179] zsig63 can also be used to identify inhibitors (antagonists) ofits activity. Test compounds are added to the assays disclosed herein toidentify compounds that inhibit the activity of zsig63. In addition tothose assays disclosed herein, samples can be tested for inhibition ofzsig63 activity within a variety of assays designed to measure receptorbinding or the stimulation/inhibition of zsig63-dependent cellularresponses. For example, zsig63-responsive cell lines can be transfectedwith a reporter gene construct that is responsive to a zsig63-stimulatedcellular pathway. Reporter gene constructs of this type are known in theart, and will generally comprise a zsig63-DNA response element operablylinked to a gene encoding an assayable protein, such as luciferase. DNAresponse elements can include, but are not limited to, cyclic AMPresponse elements (CRE), hormone response elements (HRE) insulinresponse element (IRE) (Nasrin et al., Proc. Natl. Acad. Sci. USA87:5273-7, 1990) and serum response elements (SRE) (Shaw et al. Cell56:563-72, 1989). Cyclic AMP response elements are reviewed in Roestleret al., J. Biol. Chem. 263 (19):9063-6; 1988 and Habener, Molec.Endocrinol. 4 (8):1087-94; 1990. Hormone response elements are reviewedin Beato, Cell 56:335-44; 1989. Candidate compounds, solutions, mixturesor extracts are tested for the ability to inhibit the activity of zsig63on the target cells as evidenced by a decrease in zsig63 stimulation ofreporter gene expression. Assays of this type will detect compounds thatdirectly block zsig63 binding to cell-surface receptors, as well ascompounds that block processes in the cellular pathway subsequent toreceptor-ligand binding. In the alternative, compounds or other samplescan be tested for direct blocking of zsig63 binding to receptor usingzsig63 tagged with a detectable label (e.g., 25I, biotin, horseradishperoxidase, FITC, and the like). Within assays of this type, the abilityof a test sample to inhibit the binding of labeled zsig63 to thereceptor is indicative of inhibitory activity, which can be confirmedthrough secondary assays. Receptors used within binding assays may becellular receptors or isolated, immobilized receptors.

[0180] A zsig63 polypeptide can be expressed as a fusion with animmunoglobulin heavy chain constant region, typically an Fc fragment,which contains two constant region domains and lacks the variableregion. Methods for preparing such fusions are disclosed in U.S. Pat.Nos. 5,155,027 and 5,567,584. Such fusions are typically secreted asmultimeric molecules wherein the Fc portions are disulfide bonded toeach other and two non-Ig polypeptides are arrayed in closed proximityto each other. Fusions of this type can be used to affinity purifyzsig63 receptor, as an in vitro assay tool, or as a zsig63 antagonist.For use in assays, the chimeras are bound to a support via the Fc regionand used in an ELISA format.

[0181] Zsig63 polypeptides can also be used to prepare antibodies thatbind to zsig63 epitopes, peptides or polypeptides. The zsig63polypeptide or a fragment thereof serves as an antigen (immunogen) toinoculate an animal and elicit an immune response. One of skill in theart would recognize that antigenic, epitope-bearing polypeptides containa sequence of at least 6, preferably at least 9, and more preferably atleast 15 to about 30 contiguous amino acid residues of a zsig63polypeptide (e.g., SEQ ID NO:2). Polypeptides comprising a largerportion of a zsig63 polypeptide, i.e., from 30 to 10 residues up to theentire length of the amino acid sequence are included. Antigens orimmunogenic epitopes can also include attached tags, adjuvants andcarriers, as described herein. Suitable antigens include the zsig63polypeptide encoded by SEQ ID NO:2 from amino acid number 16 (Ala) toamino acid number 219 (Gln), or a contiguous 9 to 204 amino acidfragment thereof. Other suitable antigens include domains 1, 2, and 3,and the acidic motif and coil-like region, as disclosed herein.Preferred peptides to use as antigens are hydrophilic peptides such asthose predicted by one of skill in the art from a hydrophobicity plot.Zsig63 hydrophilic peptides include peptides comprising amino acidsequences selected from the group consisting of: amino acid residues 14(Phe) to 19 (Arg) of SEQ ID NO: 2; amino acid residues 16 (Arg) to 21(Phe) of SEQ ID NO: 2; amino acid residues 24 (Gly) to 29 (Asp) of SEQID NO: 2; amino acid residues 25 (Glu) to 30 (Asp) of SEQ ID NO: 2; andamino acid residues 187Glu) to 192 (Glu) of SEQ ID NO: 2. Moreover,polypeptides that comprise hydrophilic epitopes, such as those predictedfrom a Jameson-Wolf profile, are preferred antigens, and include: aminoacid residues 24 (Gly) to 33 (Pro) of SEQ ID NO: 2; amino acid residues17 (Lys) to 33 (Pro) of SEQ ID NO: 2; amino acid residues 66 (Thr) to 73(Pro) of SEQ ID NO: 2; amino acid residues 103 (Pro) to 108 (Gly) of SEQID NO: 2; amino acid residues 190 (Ala) to 197 (Glu) of SEQ ID NO:

[0182] 2; amino acid residues 202 (Lys) to 215 (Gly) of SEQ ID NO: 2;and amino acid residues 190 (Ala) to 215 (Glu) of SEQ ID NO: 2.Antibodies from an immune response generated by inoculation of an animalwith these antigens can be isolated and purified as described herein.Methods for preparing and isolating polyclonal and monoclonal antibodiesare well known in the art. See, for example, Current Protocols inImmunology, Cooligan, et al. (eds.), National Institutes of Health, JohnWiley and Sons, Inc., 1995; Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, NY, 1989; andHurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques andApplications, CRC Press, Inc., Boca Raton, FL, 1982.

[0183] As would be evident to one of ordinary skill in the art,polyclonal antibodies can be generated from inoculating a variety ofwarm-blooded animals such as horses, cows, goats, sheep, dogs, chickens,rabbits, mice, and rats with a zsig63 polypeptide or a fragment thereof.The immunogenicity of a zsig63 polypeptide may be increased through theuse of an adjuvant, such as alum (aluminum hydroxide) or Freund'scomplete or incomplete adjuvant. Polypeptides useful for immunizationalso include fusion polypeptides, such as fusions of zsig63 or a portionthereof with an immunoglobulin polypeptide or with maltose bindingprotein. The polypeptide immunogen may be a full-length molecule or aportion thereof. If the polypeptide portion is “hapten-like”, suchportion may be advantageously joined or linked to a macromolecularcarrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin(BSA) or tetanus toxoid) for immunization.

[0184] As used herein, the term “antibodies” includes polyclonalantibodies, affinity-purified polyclonal antibodies, monoclonalantibodies, and antigen-binding fragments, such as F(ab′)₂ and Fabproteolytic fragments. Genetically engineered intact antibodies orfragments, such as chimeric antibodies, Fv fragments, single chainantibodies and the like, as well as synthetic antigen-binding peptidesand polypeptides, are also included. Non-human antibodies may behumanized by grafting non-human CDRs onto human framework and constantregions, or by incorporating the entire non-human variable domains(optionally “cloaking” them with a human-like surface by replacement ofexposed residues, wherein the result is a “veneered” antibody). In someinstances, humanized antibodies may retain non-human residues within thehuman variable region framework domains to enhance proper bindingcharacteristics. Through humanizing antibodies, biological half-life maybe increased, and the potential for adverse immune reactions uponadministration to humans is reduced. Moreover, human antibodies can beproduced in transgenic, non-human animals that have been engineered tocontain human immunoglobulin genes as disclosed in WIPO Publication WO98/24893. It is preferred that the endogenous immunoglobulin genes inthese animals be inactivated or eliminated, such as by homologousrecombination.

[0185] Antibodies are considered to be specifically binding if: 1) theyexhibit a threshold level of binding activity, and 2) they do notsignificantly cross-react with related polypeptide molecules. Athreshold level of binding is determined if anti-zsig63 antibodiesherein bind to a zsig63 polypeptide, peptide or epitope with an affinityat least 10-fold greater than the binding affinity to control(non-zsig63) polypeptide. It is preferred that the antibodies exhibit abinding affinity (K_(a)) of 10⁶ M⁻¹ or greater, preferably 10⁻¹ M⁻¹ orgreater, more preferably 10⁸ M⁻¹ or greater, and most preferably 10⁹ M⁻¹or greater. The binding affinity of an antibody can be readilydetermined by one of ordinary skill in the art, for example, byScatchard analysis (Scatchard, G., Ann. NY Acad. Sci. 51:660-672, 1949).

[0186] Whether anti-zsig63 antibodies do not significantly cross-reactwith related polypeptide molecules is shown, for example, by theantibody detecting zsig63 polypeptide but not known related polypeptidesusing a standard Western blot analysis (Ausubel et al., ibid.). Examplesof known related polypeptides are those disclosed in the prior art, suchas known orthologs, and paralogs, and similar known members of a proteinfamily, Screening can also be done using non-human zsig63, and zsig63mutant polypeptides. Moreover, antibodies can be “screened against”known related polypeptides, to isolate a population that specificallybinds to the zsig63 polypeptides. For example, antibodies raised tozsig63 are adsorbed to related polypeptides adhered to insoluble matrix;antibodies specific to zsig63 will flow through the matrix under theproper buffer conditions. Screening allows isolation of polyclonal andmonoclonal antibodies non-crossreactive to known closely relatedpolypeptides (Antibodies: A Laboratory Manual, Harlow and Lane (eds.),Cold Spring Harbor Laboratory Press, 1988; Current Protocols inImmunology, Cooligan, et al. (eds.), National Institutes of Health, JohnWiley and Sons, Inc., 1995). Screening and isolation of specificantibodies is well known in the art. See, Fundamental Immunology, Paul(eds.), Raven Press, 1993; Getzoff et al., Adv. in Immunol. 43:1-98,1988; Monoclonal Antibodies: Principles and Practice, Goding, J. W.(eds.), Academic Press Ltd., 1996; Benjamin et al., Ann. Rev. Immunol.2:67-101, 1984. Specifically binding anti-zsig63 antibodies can bedetected by a number of methods in the art, and disclosed below.

[0187] A variety of assays known to those skilled in the art can beutilized to detect antibodies which bind to zsig63 proteins orpolypeptides. Exemplary assays are described in detail in Antibodies: ALaboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor LaboratoryPress, 1988. Representative examples of such assays include: concurrentimmunoelectrophoresis, radioimmunoassay, radioimmuno-precipitation,enzyme-linked immunosorbent assay (ELISA), dot blot or Western blotassay, inhibition or competition assay, and sandwich assay. In addition,antibodies can be screened for binding to wild-type versus mutant zsig63protein or polypeptide.

[0188] Alternative techniques for generating or selecting antibodiesuseful herein include in vitro exposure of lymphocytes to zsig63 proteinor peptide, and selection of antibody display libraries in phage orsimilar vectors (for instance, through use of immobilized or labeledzsig63 protein or peptide). Genes encoding polypeptides having potentialzsig63 polypeptide binding domains can be obtained by screening randompeptide libraries displayed on phage (phage display) or on bacteria,such as E. coli. Nucleotide sequences encoding the polypeptides can beobtained in a number of ways, such as through random mutagenesis andrandom polynucleotide synthesis. These random peptide display librariescan be used to screen for peptides which interact with a known targetwhich can be a protein or polypeptide, such as a ligand or receptor, abiological or synthetic macromolecule, or organic or inorganicsubstances. Techniques for creating and screening such random peptidedisplay libraries are known in the art (Ladner et al., U.S. Pat. No.5,223,409; Ladner et al., U.S. Pat. No. 4,946,778; Ladner et al., U.S.Pat. No. 5,403,484 and Ladner et al., U.S. Pat. No. 5,571,698) andrandom peptide display libraries and kits for screening such librariesare available commercially, for instance from Clontech (Palo Alto, CA),Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc. (Beverly,Mass.) and Pharmacia LKB Biotechnology Inc. (Piscataway, N.J.). Randompeptide display libraries can be screened using the zsig63 sequencesdisclosed herein to identify proteins which bind to zsig63. These“binding polypeptides” which interact with zsig63 polypeptides can beused for tagging cells; for isolating homolog polypeptides by affinitypurification; they can be directly or indirectly conjugated to drugs,toxins, radionuclides and the like. These binding polypeptides can alsobe used in analytical methods such as for screening expression librariesand neutralizing activity, e.g., for blocking interaction between ligandand receptor, or viral binding to a receptor. The binding polypeptidescan also be used for diagnostic assays for determining circulatinglevels of zsig63 polypeptides; for detecting or quantitating solublezsig63 polypeptides as marker of underlying pathology or disease. Thesebinding polypeptides can also act as zsig63 antagonists to block zsig63binding and signal transduction in vitro and in vivo. These anti-zsig63binding polypeptides would be useful for inhibiting zsig63 activity orprotein-binding.

[0189] Antibodies to zsig63 may be used for tagging cells that expresszsig63; for isolating zsig63 by affinity purification; for diagnosticassays for determining circulating levels of zsig63 polypeptides; fordetecting or quantitating soluble zsig63 as marker of underlyingpathology or disease; in analytical methods employing FACS; forscreening expression libraries; for generating anti-idiotypicantibodies; and as neutralizing antibodies or as antagonists to blockzsig63 activity in vitro and in vivo. Suitable direct tags or labelsinclude radionuclides, enzymes, substrates, cofactors, inhibitors,fluorescent markers, chemiluminescent markers, magnetic particles andthe like; indirect tags or labels may feature use of biotin-avidin orother complement/anti-complement pairs as intermediates. Antibodiesherein may also be directly or indirectly conjugated to drugs, toxins,radionuclides and the like, and these conjugates used for in vivodiagnostic or therapeutic applications. Moreover, antibodies to zsig63or fragments thereof may be used in vitro to detect denatured zsig63 orfragments thereof in assays, for example, Western Blots or other assaysknown in the art.

[0190] Molecules of the present invention can be used to identify andisolate receptors for zsig63. For example, proteins and peptides of thepresent invention can be immobilized on a column and membranepreparations run over the column (Immobilized Affinity LigandTechniques, Hermanson et al., eds., Academic Press, San Diego, Calif.,1992, pp. 195-202). Proteins and peptides can also be radiolabeled(Methods in Enzymol., vol. 182, “Guide to Protein Purification”, M.Deutscher, ed., Acad. Press, San Diego, 1990, 721-737) or photoaffinitylabeled (Brunner et al., Ann. Rev. Biochem. 62:483-514, 1993 and Fedanet al., Biochem. Pharmacol. 33:1167-1180, 1984) and specificcell-surface proteins can be identified.

[0191] Differentiation is a progressive and dynamic process, beginningwith pluripotent stem cells and ending with terminally differentiatedcells. Pluripotent stem cells that can regenerate without commitment toa lineage express a set of differentiation markers that are lost whencommitment to a cell lineage is made. Progenitor cells express a set ofdifferentiation markers that may or may not continue to be expressed asthe cells progress down the cell lineage pathway toward maturation.Differentiation markers that are expressed exclusively by mature cellsare usually functional properties such as cell products, enzymes toproduce cell products, and receptors. The stage of a cell population'sdifferentiation is monitored by identification of markers present in thecell population. Myocytes, osteoblasts, adipocytes, chrondrocytes,fibroblasts and reticular cells are believed to originate from a commonmesenchymal stem cell (Owen et al., Ciba Fdn. Symp. 136:42-46, 1988).Markers for mesenchymal stem cells have not been well identified (Owenet al., J. of Cell Sci. 87:731-738, 1987), so identification is usuallymade at the progenitor and mature cell stages. The novel polypeptides ofthe present invention may be useful for studies to isolate mesenchymalstem cells and myocyte or other progenitor cells, both in vivo and exvivo.

[0192] There is evidence to suggest that factors that stimulate specificcell types down a pathway towards terminal differentiation ordedifferentiation affect the entire cell population originating from acommon precursor or stem cell. Thus, the present invention includesstimulating or inhibiting the proliferation of myocytes, smooth musclecells, osteoblasts, adipocytes, chrondrocytes, neuronal and endothelialcells. Molecules of the present invention for example, may whilestimulating proliferation or differentiation of cardiac myocytes,inhibit proliferation or differentiation of adipocytes, by virtue of theaffect on their common precursor/stem cells. Thus molecules of thepresent invention may have use in inhibiting chondrosarcomas,atherosclerosis, restenosis and obesity.

[0193] Assays measuring differentiation include, for example, measuringcell markers associated with stage-specific expression of a tissue,enzymatic activity, functional activity or morphological changes (Watt,FASEB, 5:281-284, 1991; Francis, Differentiation 57:63-75, 1994; Raes,Adv. Anim. Cell Biol. Technol. Bioprocesses, 161-171, 1989; allincorporated herein by reference). Alternatively, zsig63 polypeptideitself can serve as an additional cell-surface or secreted markerassociated with stage-specific expression of a tissue. As such, directmeasurement of zsig63 polypeptide, or its loss of expression in a tissueas it differentiates, can serve as a marker for differentiation oftissues.

[0194] Similarly, direct measurement of zsig63 polypeptide, or its lossof expression in a tissue can be determined in a tissue or cells as theyundergo tumor progression. Increases in invasiveness and motility ofcells, or the gain or loss of expression of zsig63 in a pre-cancerous orcancerous condition, in comparison to normal tissue, can serve as adiagnostic for transformation, invasion and metastasis in tumorprogression. As such, knowledge of a tumor's stage of progression ormetastasis will aid the physician in choosing the most proper therapy,or aggressiveness of treatment, for a given individual cancer patient.Methods of measuring gain and loss of expression (of either mRNA orprotein) are well known in the art and described herein and can beapplied to zsig63 expression. For example, appearance or disappearanceof polypeptides that regulate cell motility can be used to aid diagnosisand prognosis of prostate cancer (Banyard, J. and Zetter, B. R., Cancerand Metast. Rev. 17:449-458, 1999). As an effector of cell motility,zsig63 gain or loss of expression may serve as a diagnostic for prostateand other cancers.

[0195] Moreover, the activity and effect of zsig63 on tumor progressionand metastasis can be measured in vivo. Several syngeneic mouse modelshave been developed to study the influence of polypeptides, compounds orother treatments on tumor progression. In these models, tumor cellspassaged in culture are implanted into mice of the same strain as thetumor donor. The cells will develop into tumors having similarcharacteristics in the recipient mice, and metastasis will also occur insome of the models. Appropriate tumor models for our studies include theLewis lung carcinoma (ATCC No. CRL-1642) and B16 melanoma (ATCC No.CRL-6323), amongst others. These are both commonly used tumor lines,syngeneic to the C57BL6 mouse, that are readily cultured and manipulatedin vitro. Tumors resulting from implantation of either of these celllines are capable of metastasis to the lung in C57BL6 mice. The Lewislung carcinoma model has recently been used in mice to identify aninhibitor of angiogenesis (O'Reilly MS, et al. Cell 79:315-328,1994).C57BL6/J mice are treated with an experimental agent either throughdaily injection of recombinant protein, agonist or antagonist or a onetime injection of recombinant adenovirus. Three days following thistreatment, 10⁵ to 10⁶ cells are implanted under the dorsal skin.Alternatively, the cells themselves may be infected with recombinantadenovirus, such as one expressing zsig63, before implantation so thatthe protein is synthesized at the tumor site or intracellularly, ratherthan systemically. The mice normally develop visible tumors within 5days. The tumors are allowed to grow for a period of up to 3 weeks,during which time they may reach a size of 1500 -1800 mm³ in the controltreated group. Tumor size and body weight are carefully monitoredthroughout the experiment. At the time of sacrifice, the tumor isremoved and weighed along with the lungs and the liver. The lung weighthas been shown to correlate well with metastatic tumor burden. As anadditional measure, lung surface metastases are counted. The resectedtumor, lungs and liver are prepared for histopathological examination,immunohistochemistry, and in situ hybridization, using methods known inthe art and described herein. The influence of the expressed polypeptidein question, e.g., zsig63, on the ability of the tumor to recruitvasculature and undergo metastasis can thus be assessed. In addition,aside from using adenovirus, the implanted cells can be transientlytransfected with zsig63. Use of stable zsig63 transfectants as well asuse of induceable promoters to activate zsig63 expression in vivo areknown in the art and can be used in this system to assess zsig63induction of metastasis. Moreover, purified zsig63 or zsig63 conditionedmedia can be directly injected in to this mouse model, and hence be usedin this system. For general reference see, O'Reilly MS, et al. Cell79:315-328, 1994; and Rusciano D, et al. Murine Models of LiverMetastasis. Invasion Metastasis 14:349-361, 1995.

[0196] The activity of zsig63 and its derivatives (conjugates) on growthand dissemination of tumor cells derived from human hematologicmalignancies can also be measured in vivo in a mouse Xenograft modelSeveral mouse models have been developed in which human tumor cells areimplanted into immunodeficient mice, collectively referred to asxenograft models. See Cattan, AR and Douglas, E Leuk. Res. 18:513-22,1994; and Flavell, DJ, Hematological Oncology 14:67-82, 1996. Thecharacteristics of the disease model vary with the type and quantity ofcells delivered to the mouse. Typically, the tumor cells willproliferate rapidly and can be found circulating in the blood andpopulating numerous organ systems. Therapeutic strategies appropriatefor testing in such a model include antibody induced toxicity,ligand-toxin conjugates or cell-based therapies. The latter method,commonly referred to adoptive immunotherapy, involves treatment of theanimal with components of the human immune system (i.e. lymphocytes, NKcells) and may include ex vivo incubation of cells with zsig63 or otherimmunomodulatory agents.

[0197] Polynucleotides encoding zsig63 polypeptides are useful withingene therapy applications where it is desired to increase or inhibitzsig63 activity. If a mammal has a mutated or absent zsig63 gene, thezsig63 gene can be introduced into the cells of the mammal. In oneembodiment, a gene encoding a zsig63 polypeptide is introduced in vivoin a viral vector. Such vectors include an attenuated or defective DNAvirus, such as, but not limited to, herpes simplex virus (HSV),papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associatedvirus (AAV), and the like. Defective viruses, which entirely or almostentirely lack viral genes, are preferred. A defective virus is notinfective after introduction into a cell. Use of defective viral vectorsallows for administration to cells in a specific, localized area,without concern that the vector can infect other cells. Examples ofparticular vectors include, but are not limited to, a defective herpessimplex virus I (HSV1) vector (Kaplitt et al., Molec. Cell. Neurosci.2:320-30, 1991); an attenuated adenovirus vector, such as the vectordescribed by Stratford-Perricaudet et al., J. Clin. Invest. 90:626-30,1992; and a defective adeno-associated virus vector (Samulski et al., J.Virol. 61:3096-101, 1987; Samulski et al., J. Virol. 63:3822-8, 1989).

[0198] In another embodiment, a zsig63 gene can be introduced in aretroviral vector, e.g., as described in Anderson et al., U.S. Pat. No.5,399,346; Mann et al. Cell 33:153, 1983; Temin et al., U.S. Pat. No.4,650,764; Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., J.Virol. 62:1120, 1988; Temin et al., U.S. Pat. No. 5,124,263;International Patent Publication No. WO 95/07358, published Mar. 16,1995 by Dougherty et al.; and Kuo et al., Blood 82:845, 1993.Alternatively, the vector can be introduced by lipofection in vivo usingliposomes. Synthetic cationic lipids can be used to prepare liposomesfor in vivo transfection of a gene encoding a marker (Felgner et al.,Proc. Natl. Acad. Sci. USA 84:7413-7, 1987; Mackey et al., Proc. Natl.Acad. Sci. USA 85:8027-31, 1988). The use of lipofection to introduceexogenous genes into specific organs in vivo has certain practicaladvantages. Molecular targeting of liposomes to specific cellsrepresents one area of benefit. More particularly, directingtransfection to particular cells represents one area of benefit. Forinstance, directing transfection to particular cell types would beparticularly advantageous in a tissue with cellular heterogeneity, suchas the pancreas, liver, kidney, and brain. Lipids may be chemicallycoupled to other molecules for the purpose of targeting. Targetedpeptides (e.g., hormones or neurotransmitters), proteins such asantibodies, or non-peptide molecules can be coupled to liposomeschemically.

[0199] It is possible to remove the target cells from the body; tointroduce the vector as a naked DNA plasmid; and then to re-implant thetransformed cells into the body. Naked DNA vectors for gene therapy canbe introduced into the desired host cells by methods known in the art,e.g., transfection, electroporation, microinjection, transduction, cellfusion, DEAE dextran, calcium phosphate precipitation, use of a gene gunor use of a DNA vector transporter. See, e.g., Wu et al., J. Biol. Chem.267:963-7, 1992; Wu et al., J. Biol. Chem. 263:14621-4, 1988.

[0200] Antisense methodology can be used to inhibit zsig63 gene 25transcription, such as to inhibit cell proliferation in vivo.Polynucleotides that are complementary to a segment of a zsig63-encodingpolynucleotide (e.g., a polynucleotide as set forth in SEQ ID NO:1) aredesigned to bind to zsig63-encoding mRNA and to inhibit translation ofsuch mRNA. Such antisense polynucleotides are used to inhibit expressionof zsig63 polypeptide-encoding genes in cell culture or in a subject.

[0201] Polynucleotides of the present invention are also used to detectabnormalities on human chromosome 4 associated with disease or otherhuman traits. The polynucleotides of the present invention map to the4q12-4q13region on human chromosome 4. Zsig63 maps 3.15 cR_(—)3000 fromthe framework marker WI-7844 on the chromosome 4 WICGR radiation hybridmap. Proximal and distal framework markers were WI-7844 and WI-4767,respectively. The use of surrounding markers positions zsig63 in the4q12-4q13 region on the integrated LDB chromosome 4 map. (see Example3).

[0202] The present invention also provides reagents which will find usein diagnostic applications. For example, the zsig63 gene, a probecomprising zsig63 DNA or RNA or a subsequence thereof can be used todetermine if the zsig63 gene is present on chromosome 4 or if a mutationhas occurred. Detectable chromosomal aberrations at the zsig63 genelocus include but are not limited to aneuploidy, gene copy numberchanges, insertions, deletions, translocations, restriction site changesand rearrangements. Such aberrations can be detected usingpolynucleotides of the present invention by employing molecular genetictechniques, such as restriction fragment length polymorphism (RFLP)analysis, short tandem repeat (STR) analysis employing PCR techniques,and other genetic linkage analysis techniques known in the art (Sambrooket al., ^(ibid).; Ausubel, et. al., ^(ibid).; Marian, A. J., ^(Chest,)108:255-265, 1995). These methods can be employed to use zsig63polynucleotides to detect abnormalities on human chromosome 4, such asthose described below.

[0203] The precise knowledge of a gene's position can be useful for anumber of purposes, including: 1) determining if a sequence is part ofan existing contig and obtaining additional surrounding geneticsequences in various forms, such as YACs, BACs or cDNA clones; 2)providing a possible candidate gene for an inheritable disease whichshows linkage to the same chromosomal region; and 3) cross-referencingmodel organisms, such as mouse, which may aid in determining whatfunction a particular gene has.

[0204] Zsig63 is located at the 4q12-4q13 region of chromosome 4.Several genes of known function map to this region. Zsig63polynucleotide probes can be used to detect abnormalities or genotypesassociated with these defects. There are several genes that map to thezsig63 locus that are associated with human disease states, such asdental diseases: For example, Dentinogenesis imperfecta (4q13-q21) anddentin dysplasia, type II (4q) map near zsig63. As a salivary proteinthat also maps to this region of chromosome 4, defects in zsig63 mayalso play a role in dental disease, such as causing a defect in dentalformation; and/or predisposition to dental carries and periodontaldisease, as the saliva is important in maintaining a healthy microbialenvironment in the mouth. Moreover, a v-kit oncogene homolog related toPiebaldism and certain severe malignancies (e.g., certain leukemias)maps to 4ql2 and is involved in common malignant gastrointestinalstromal tumors (GIST) tumors. As zsig63 maps to 4q12-q13 as well, therecan be an association between loss of zsig63 function and tumorformation or progression. Moreover, translocations and duplications in4q12 are seen in v-kit oncogene homolog related diseases. Thus, zsig63polynucleotide probes can be used to detect abnormalities or genotypesassociated with these diseases cancer susceptibility markers, as well asdetection of chromosome translocations associated therewith. Becausethere is abundant evidence for cancer resulting from mutations in the4q12 region, and zsig63 also maps to this chromosomal locus, mutationsin zsig63 may also be directly involved in or associated with cancers,such as lymphoid cell cancers, gastrointestinal and salivary glandtumors, solid tumors or other tumors.

[0205] A diagnostic could assist physicians in determining the type ofdisease and appropriate associated therapy, or assistance in geneticcounseling. As such, the inventive anti-zsig63 antibodies,polynucleotides, and polypeptides can be used for the detection ofzsig63 polypeptide, mRNA or anti-zsig63 antibodies, thus serving asmarkers and be directly used for detecting or genetic diseases orcancers, as described herein, using methods known in the art anddescribed herein. Further, zsig63 polynucleotide probes can be used todetect abnormalities or genotypes associated with chromosome 4q12-q13deletions and translocations associated with human diseases, such asthose described above, or other translocations involved with malignantprogression of tumors or other 4q12-q13 mutations, which are expected tobe involved in chromosome rearrangements in malignancy; or in othercancers. Similarly, zsig63 polynucleotide probes can be used to detectabnormalities or genotypes associated with chromosome 4q12-q13 trisomyand chromosome loss associated with human diseases or spontaneousabortion. Moreover, amongst other genetic loci, those for Sarcoglycanlinked muscular dystrophy (4ql2), Stargardt Disease (4q), and others,all manifest themselves in human disease states as well as map to thisregion of the human genome. See the Online Mendellian Inheritance of Man(OMIM) gene map, and references therein, for this region of chromosome 4on a publicly available WWW server(http://www3.ncbi.nlm.nih.gov/htbin-post/Omim/getmap?chromosome=4q12).All of these serve as possible candidate genes for an inheritabledisease which show linkage to the same chromosomal region the zsig63gene. Thus, zsig63 polynucleotide probes can be used to detectabnormalities or genotypes associated with these defects.

[0206] A diagnostic could assist physicians in determining the type ofdisease and appropriate associated therapy, or assistance in geneticcounseling. As such, the inventive anti-zsig63 antibodies,polynucleotides, and polypeptides can be used for the detection ofzsig63 polypeptide, mRNA or anti-zsig63 antibodies, thus serving asmarkers and be directly used for detecting or genetic diseases orcancers, as described herein, using methods known in the art anddescribed herein. Further, zsig63 polynucleotide probes can be used todetect abnormalities or genotypes associated with chromosome 4q12-q13deletions and translocations associated with human diseases, or othertranslocations involved with malignant progression of tumors or other4q12-q13 mutations, which are expected to be involved in chromosomerearrangements in malignancy; or in other cancers. Similarly, zsig63polynucleotide probes can be used to detect abnormalities or genotypesassociated with chromosome 4q12-q13 trisomy and chromosome lossassociated with human diseases or spontaneous abortion. Thus, zsig63polynucleotide probes can be used to detect abnormalities or genotypesassociated with these defects.

[0207] Similarly, defects in the zsig63 gene itself may result in aheritable human disease state. Molecules of the present invention, suchas the polypeptides, antagonists, agonists, polynucleotides andantibodies of the present invention would aid in the detection,diagnosis prevention, and treatment associated with a zsig63 geneticdefect. As discussed above, defects in the zsig63 gene itself may resultin a heritable human disease state or increase susceptibility to certaindiseases. Molecules of the present invention, such as the polypeptides,antagonists, agonists, polynucleotides and antibodies of the presentinvention would aid in the detection, diagnosis prevention, andtreatment associated with a zsig63 genetic defect. In addition, zsig63polynucleotide probes can be used to detect allelic differences betweendiseased or non-diseased individuals at the zsig63 chromosomal locus. Assuch, the zsig63 sequences can be used as diagnostics in forensic DNAprofiling.

[0208] In general, the diagnostic methods used in genetic linkageanalysis, to detect a genetic abnormality or aberration in a patient,are known in the art. Most diagnostic methods comprise the steps of (a)obtaining a genetic sample from a potentially diseased patient, diseasedpatient or potential non-diseased carrier of a recessive disease allele;(b) producing a first reaction product by incubating the genetic samplewith a zsig63 polynucleotide probe wherein the polynucleotide willhybridize to complementary polynucleotide sequence, such as in RFLPanalysis or by incubating the genetic sample with sense and antisenseprimers in a PCR reaction under appropriate PCR reaction conditions;(iii) Visualizing the first reaction product by gel electrophoresisand/or other known method such as visualizing the first reaction productwith a zsig63 polynucleotide probe wherein the polynucleotide willhybridize to the complementary polynucleotide sequence of the firstreaction; and (iv) comparing the visualized first reaction product to asecond control reaction product of a genetic sample from wild typepatient. A difference between the first reaction product and the controlreaction product is indicative of a genetic abnormality in the diseasedor potentially diseased patient, or the presence of a heterozygousrecessive carrier phenotype for a non-diseased patient, or the presenceof a genetic defect in a tumor from a diseased patient, or the presenceof a genetic abnormality in a fetus or pre-implantation embryo. Forexample, a difference in restriction fragment pattern, length of PCRproducts, length of repetitive sequences at the zsig63 genetic locus,and the like, are indicative of a genetic abnormality, geneticaberration, or allelic difference in comparison to the normal wild typecontrol. Controls can be from unaffected family members, or unrelatedindividuals, depending on the test and availability of samples. Geneticsamples for use within the present invention include genomic DNA, mRNA,and cDNA isolated form any tissue or other biological sample from apatient, such as but not limited to, blood, saliva, semen, embryoniccells, amniotic fluid, and the like. The polynucleotide probe or primercan be RNA or DNA, and will comprise a portion of SEQ ID NO: 1, thecomplement of SEQ ID NO: 1, or an RNA equivalent thereof. Such methodsof showing genetic linkage analysis to human disease phenotypes are wellknown in the art. For reference to PCR based methods in diagnostics seesee, generally, Mathew (ed.), Protocols in Human Molecular Genetics(Humana Press, Inc. 1991), White (ed.), PCR Protocols: Current Methodsand Applications (Humana Press, Inc. 1993), Cotter (ed.), MolecularDiagnosis of Cancer (Humana Press, Inc. 1996), Hanausek and Walaszek(eds.), Tumor Marker Protocols (Humana Press, Inc. 1998), Lo (ed.),Clinical Applications of PCR (Humana Press, Inc. 1998), and Meltzer(ed.), PCR in Bioanalysis (Humana Press, Inc. 1998)).

[0209] Mutations associated with the zsig63 locus can be detected usingnucleic acid molecules of the present invention by employing standardmethods for direct mutation analysis, such as restriction fragmentlength polymorphism analysis, short tandem repeat analysis employing PCRtechniques, amplification-refractory mutation system analysis,single-strand conformation polymorphism detection, RNase cleavagemethods, denaturing gradient gel electrophoresis, fluorescence-assistedmismatch analysis, and other genetic analysis techniques known in theart (see, for example, Mathew (ed.), Protocols in Human MolecularGenetics (Humana Press, Inc. 1991), Marian, Chest 108:255 (1995),Coleman and Tsongalis, Molecular Diagnostics (Human Press, Inc. 1996),Elles (ed.) Molecular Diagnosis of Genetic Diseases (Humana Press, Inc.1996), Landegren (ed.), Laboratory Protocols for Mutation Detection(Oxford University Press 1996), Birren et al. (eds.), Genome Analysis,Vol. 2: Detecting Genes (Cold Spring Harbor Laboratory Press 1998),Dracopoli et al. (eds.), Current Protocols in Human Genetics (John Wiley& Sons 1998), and Richards and Ward, “Molecular Diagnostic Testing,” inPrinciples of Molecular Medicine, pages 83-88 (Humana Press, Inc.1998)). Direct analysis of an zsig63 gene for a mutation can beperformed using a subject's genomic DNA.

[0210] Methods for amplifying genomic DNA, obtained for example fromperipheral blood lymphocytes, are well-known to those of skill in theart (see, for example, Dracopoli et al. (eds.), Current Protocols inHuman Genetics, at pages 7.1.6 to 7.1.7 (John Wiley & Sons 1998)).

[0211] Mice engineered to express the zsig63 gene, referred to as“transgenic mice,” and mice that exhibit a complete absence of zsig63gene function, referred to as “knockout mice,” may also be generated(Snouwaert et al., Science 257:1083, 1992; Lowell et al., Nature366:740-42, 1993; Capecchi, M. R., Science 244:1288-1292, 1989;Palmiter, R. D. et al. Annu Rev Genet. 20:465-499, 1986). For example,transgenic mice that over-express zsig63, either ubiquitously or under atissue-specific or tissue-restricted promoter can be used to ask whetherover-expression causes a phenotype. For example, over-expression of awild-type zsig63 polypeptide, polypeptide fragment or a mutant thereofmay alter normal cellular processes, resulting in a phenotype thatidentifies a tissue in which zsig63 expression is functionally relevantand may indicate a therapeutic target for the zsig63, its agonists orantagonists. For example, a preferred transgenic mouse to engineer isone that over-expresses the zsig63 mature polypeptide (approximatelyamino acids 16 (Ala) to 219 (Gln) of SEQ ID NO:2). Moreover, suchover-expression may result in a phenotype that shows similarity withhuman diseases. Similarly, knockout zsig63 mice can be used to determinewhere zsig63 is absolutely required in vivo. The phenotype of knockoutmice is predictive of the in vivo effects of that a zsig63 antagonist,such as those described herein, may have. The human zsig63 cDNA can beused to isolate murine zsig63 mRNA, cDNA and genomic DNA, which aresubsequently used to generate knockout mice. These mice may be employedto study the zsig63 gene and the protein encoded thereby in an in vivosystem, and can be used as in vivo models for corresponding humandiseases. Moreover, transgenic mice expression of zsig63 antisensepolynucleotides or ribozymes directed against zsig63, described herein,can be used analogously to transgenic mice described above.

[0212] For pharmaceutical use, the proteins of the present invention areformulated for topical, inhalant or parenteral, particularly intravenousor subcutaneous, delivery according to conventional methods. Intravenousadministration will be by bolus injection or infusion over a typicalperiod of one to several hours. In general, pharmaceutical formulationswill include a zsig63 protein in combination with a pharmaceuticallyacceptable vehicle, such as saline, buffered saline, 5% dextrose inwater or the like. Formulations may further include one or moreexcipients, preservatives, solubilizers, buffering agents, albumin toprevent protein loss on vial surfaces, etc. Methods of formulation arewell known in the art and are disclosed, for example, in Remington: TheScience and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co.,Easton, PA, 19th ed., 1995. Therapeutic doses will generally determinedby the clinician according to accepted standards, taking into accountthe nature and severity of the condition to be treated, patient traits,etc. Determination of dose is within the level of ordinary skill in theart. The proteins may be administered for acute treatment, over one weekor less, often over a period of one to three days or may be used inchronic treatment, over several months or years.

[0213] The invention is further illustrated by the followingnon-limiting examples.

EXAMPLE 1 Identification of zsig63 Using an EST Sequence to ObtainFull-length zsig63

[0214] Scanning of a translated DNA database resulted in identificationof an expressed sequence tag (EST) sequence found to be a novel memberof the adhesin family and designated zsig63.

[0215] Confirmation of the EST sequence was made by sequence analyses of25 the cDNA from which the EST originated. This cDNA clone was obtainedand sequenced using the following primers: ZC6768 (SEQ ID NO:4), ZC694(SEQ ID NO:5), ZC7231 (SEQ ID NO:6), ZC7764a (SEQ ID NO:7). The insertwas about 1 kb and was full-length.

EXAMPLE 2 Tissue Distribution

[0216] Northern blot analysis was performed using Human Multiple TissueNorthern™ Blots (MTN I, MTN II, MTN III) (Clontech) and an in-houseNorthern blot prepared using 2 mg each of human testis, prostate spinalcord, salivary gland, thymus, and thyroid poly-A+RNA (Clontech). Theplasmid containing full-length zsig63 (Example 1) was digested as permanufacturer's instructions with HindlIl and Xbal (New England BioLabs,Beverly, MA) for 2 hours at 37° C. A sample of the digested plasmid DNAwas run on a 1% agarose gel. A band of the expected size of 662 bp wasseen. The 662 bp fragment, was gel purified using a commerciallyavailable kit (QiaexII™; Qiagen) and then radioactively labeled with³²P-dCTP using Rediprime II™ (Amersham), a random prime labeling system,according to the manufacturer's specifications. The probe was thenpurified using a Nuc-Trap™ column (Stratagene) according to themanufacturer's instructions. ExpressHyb™ (Clontech) solution was usedfor prehybridization and as a hybridizing solution for the Northernblots. Hybridization took place overnight at 65° C. using 1-2×10⁶ cpm/mlof labeled probe. The blots were then washed 4 times for 20 minutes in2X SSC/0.1% SDS at 25° C., and then twice more in 0.1 X SSC/0.1% SDS at50° C. for 30 minutes each. A transcript of approximately 1.3 kb wasdetected at very high levels in salivary gland, and moderate to lowlevels in thyroid and prostate.

[0217] Dot Blots were also performed using Human RNA Master Blots™(Clontech). The methods and conditions for the Dot Blots are the same asfor the Multiple Tissue Blots described above. Dot blot had a verystrong signal in salivary gland.

EXAMPLE 3 Chromosomal Mapping of the zsig63 Gene

[0218] Zsig63 was mapped to chromosome 4 using the commerciallyavailable “GeneBridge 4 Radiation Hybrid Panel” (Research Genetics,Inc., Huntsville, Ala.). The GeneBridge 4 Radiation Hybrid Panelcontains DNAs from each of 93 radiation hybrid clones, plus two controlDNAs (the HFL donor and the A23 recipient). A publicly available WWWserver (http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl) allowsmapping relative to the Whitehead Institute/MIT Center for GenomeResearch's radiation hybrid map of the human genome (the “WICGR”radiation hybrid map) which was constructed with the GeneBridge 4Radiation Hybrid Panel.

[0219] For the mapping of zsig63 with the “GeneBridge 4 RH Panel”, 20 μlreactions were set up in a 96-well microtiter plate (Stratagene, LaJolla, Calif.) and used in a “RoboCycler Gradient 96” thermal cycler(Stratagene). Each of the 95 PCR reactions consisted of 2 μl 10 XKlenTaq PCR reaction buffer (Clontech Laboratories, Inc., Palo Alto,Calif.), 1.6 μl dNTPs mix (2.5 mM each, Perkin-Elmer, Foster City,Calif.), 1 μl sense primer, ZC 20,555, (SEQ ID NO:8) 1 μl antisenseprimer, ZC 20,556, (SEQ ID NO:9) 2 μl “RediLoad” (Research Genetics,Inc., Huntsville, Ala.), 0.4 μl 50 X Advantage KlenTaq Polymerase Mix(Clontech), 25 ng of DNA from an individual hybrid clone or control andddH₂O for a total volume of 20 μl. The reactions were overlaid with anequal amount of mineral oil and sealed. The PCR cycler conditions wereas follows: an initial 1 cycle 5 minute denaturation at 94° C., 35cycles of a 45 seconds denaturation at 94° C., 45 seconds annealing at58° C. and 1 minute and 15 seconds extension at 72° C., followed by afinal 1 cycle extension of 7 minutes at 72° C. The reactions wereseparated by electrophoresis on a 2% agarose gel (Life Technologies,Gaithersburg, Md.).

[0220] The results showed that zsig63 maps 3.15 cR_(—)3000 from theframework marker WI-7844 on the chromosome 4 WICGR radiation hybrid map.Proximal and distal framework markers were WI-7844 and WI-4767,respectively. The use of surrounding markers positions zsig63 in the4ql2-4ql3 region on the integrated LDB chromosome 4 map (The GeneticLocation Database, University of Southhampton, WWW server: http://cedar. genetics. soton.ac.uk/public_html/).

[0221] From the foregoing, it will be appreciated that, althoughspecific embodiments of the invention have been described herein forpurposes of illustration, various modifications may be made withoutdeviating from the spirit and scope of the invention. Accordingly, theinvention is not limited except as by the appended claims.

1 9 1 1008 DNA Homo sapiens CDS (128)...(784) 1 agacagacta aaaaagccatgtattctttc gtttctctct aaaagaagaa aaatataatt 60 taaaaataca ttgcgtattttctaaaacaa taaatttata gtgttaatat tcatagggtc 120 aatcaaa atg aag ctt ctcctt tgg gcc tgc att gta tgt gtt gct ttt 169 Met Lys Leu Leu Leu Trp AlaCys Ile Val Cys Val Ala Phe 1 5 10 gca agg aag aga cgg ttc ccc ttc attggt gag gat gac aat gac gat 217 Ala Arg Lys Arg Arg Phe Pro Phe Ile GlyGlu Asp Asp Asn Asp Asp 15 20 25 30 ggt cac cca ctt cat cca tct ctg aatatt cct tat ggc ata cgg aat 265 Gly His Pro Leu His Pro Ser Leu Asn IlePro Tyr Gly Ile Arg Asn 35 40 45 tta cca cct cct ctt tat tat cgc cca gtgaat aca gtc ccc agt tac 313 Leu Pro Pro Pro Leu Tyr Tyr Arg Pro Val AsnThr Val Pro Ser Tyr 50 55 60 cct ggg aat act tac act gac aca ggg tta ccttcg tat ccc tgg att 361 Pro Gly Asn Thr Tyr Thr Asp Thr Gly Leu Pro SerTyr Pro Trp Ile 65 70 75 cta act tct cct gga ttc ccc tat gtc tat cac atccgt ggt ttt ccc 409 Leu Thr Ser Pro Gly Phe Pro Tyr Val Tyr His Ile ArgGly Phe Pro 80 85 90 tta gct act cag ttg aat gtt cct cct ctc cct cct aggggt ttc ccg 457 Leu Ala Thr Gln Leu Asn Val Pro Pro Leu Pro Pro Arg GlyPhe Pro 95 100 105 110 ttt gtc cct cct tca agg ttt ttt tca gca gct gcagca ccc gct gcc 505 Phe Val Pro Pro Ser Arg Phe Phe Ser Ala Ala Ala AlaPro Ala Ala 115 120 125 cca cct att gca gct gag cct gct gca gct gca cctctt aca gcc aca 553 Pro Pro Ile Ala Ala Glu Pro Ala Ala Ala Ala Pro LeuThr Ala Thr 130 135 140 cct gta gca gct gag cct gct gca ggg gcc cct gttgca gct gag cct 601 Pro Val Ala Ala Glu Pro Ala Ala Gly Ala Pro Val AlaAla Glu Pro 145 150 155 gct gca gag gca cct gtt gga gct gag cct gct gcagag gca cct gtt 649 Ala Ala Glu Ala Pro Val Gly Ala Glu Pro Ala Ala GluAla Pro Val 160 165 170 gca gct gag cct gct gca gag gca cct gtt gga gtggag cca gct gca 697 Ala Ala Glu Pro Ala Ala Glu Ala Pro Val Gly Val GluPro Ala Ala 175 180 185 190 gag gaa cct tca cca gct gag cct gct aca gccaag cct gct gcc cca 745 Glu Glu Pro Ser Pro Ala Glu Pro Ala Thr Ala LysPro Ala Ala Pro 195 200 205 gaa cct cac cct tct ccc tct ctt gaa cag gcaaat cag tgaaattctc 794 Glu Pro His Pro Ser Pro Ser Leu Glu Gln Ala AsnGln 210 215 tagaagagta ccatgggttc atttctatac tgatgcagaa ataagtgaaatctacaaaag 854 ttttctttct tttccaaaga ctatttcatt ctgttgtatt cagagtattcatctcactac 914 attgatttgt ttgtggtagt ttttccttgg acttaattta tattgaaaaaacattgataa 974 ttaaataaat aaaatagata atttagacca atgg 1008 2 219 PRT Homosapiens 2 Met Lys Leu Leu Leu Trp Ala Cys Ile Val Cys Val Ala Phe AlaArg 1 5 10 15 Lys Arg Arg Phe Pro Phe Ile Gly Glu Asp Asp Asn Asp AspGly His 20 25 30 Pro Leu His Pro Ser Leu Asn Ile Pro Tyr Gly Ile Arg AsnLeu Pro 35 40 45 Pro Pro Leu Tyr Tyr Arg Pro Val Asn Thr Val Pro Ser TyrPro Gly 50 55 60 Asn Thr Tyr Thr Asp Thr Gly Leu Pro Ser Tyr Pro Trp IleLeu Thr 65 70 75 80 Ser Pro Gly Phe Pro Tyr Val Tyr His Ile Arg Gly PhePro Leu Ala 85 90 95 Thr Gln Leu Asn Val Pro Pro Leu Pro Pro Arg Gly PhePro Phe Val 100 105 110 Pro Pro Ser Arg Phe Phe Ser Ala Ala Ala Ala ProAla Ala Pro Pro 115 120 125 Ile Ala Ala Glu Pro Ala Ala Ala Ala Pro LeuThr Ala Thr Pro Val 130 135 140 Ala Ala Glu Pro Ala Ala Gly Ala Pro ValAla Ala Glu Pro Ala Ala 145 150 155 160 Glu Ala Pro Val Gly Ala Glu ProAla Ala Glu Ala Pro Val Ala Ala 165 170 175 Glu Pro Ala Ala Glu Ala ProVal Gly Val Glu Pro Ala Ala Glu Glu 180 185 190 Pro Ser Pro Ala Glu ProAla Thr Ala Lys Pro Ala Ala Pro Glu Pro 195 200 205 His Pro Ser Pro SerLeu Glu Gln Ala Asn Gln 210 215 3 657 DNA Artificial Sequence Degeneratepolynucleotide sequence for zsig63 3 atgaarytny tnytntgggc ntgyathgtntgygtngcnt tygcnmgnaa rmgnmgntty 60 ccnttyathg gngargayga yaaygaygayggncayccny tncayccnws nytnaayath 120 ccntayggna thmgnaayyt nccnccnccnytntaytaym gnccngtnaa yacngtnccn 180 wsntayccng gnaayacnta yacngayacnggnytnccnw sntayccntg gathytnacn 240 wsnccnggnt tyccntaygt ntaycayathmgnggnttyc cnytngcnac ncarytnaay 300 gtnccnccny tnccnccnmg nggnttyccnttygtnccnc cnwsnmgntt yttywsngcn 360 gcngcngcnc cngcngcncc nccnathgcngcngarccng cngcngcngc nccnytnacn 420 gcnacnccng tngcngcnga rccngcngcnggngcnccng tngcngcnga rccngcngcn 480 gargcnccng tnggngcnga rccngcngcngargcnccng tngcngcnga rccngcngcn 540 gargcnccng tnggngtnga rccngcngcngargarccnw snccngcnga rccngcnacn 600 gcnaarccng cngcnccnga rccncayccnwsnccnwsny tngarcargc naaycar 657 4 25 DNA Artificial SequenceOligonucleotide primer ZC6768 4 gcaattaacc ctcactaaag ggaac 25 5 20 DNAArtificial Sequence Oligonucleotide primer ZC694 5 taatacgact cactataggg20 6 26 DNA Artificial Sequence Oligonucleotide primer ZC7231 6tttttttttt tttttttttt tttttv 26 7 26 DNA Artificial SequenceOligonucleotide primer ZC7764a 7 tttttttttt tttttttttt ttttta 26 8 18DNA Artificial Sequence Oligonucleotide primer ZC20555 8 ccacctcctctttattat 18 9 18 DNA Artificial Sequence Oligonucleotide primer ZC205569 aatccaggag aagttaga 18

What is claimed is:
 1. An isolated polynucleotide encoding a zsig63polypeptide comprising a sequence of amino acid residues that is atleast 90% identical to an amino acid sequence selected from the groupconsisting of: (a) the amino acid sequence as shown in SEQ ID NO: 2 fromamino acid number 16 (Arg) to amino acid number 37 (Ser); (b) the aminoacid sequence as shown in SEQ ID NO: 2 from amino acid number 38 (Leu)to amino acid number 126 (Ala); (c) the amino acid sequence as shown inSEQ ID NO: 2 from amino acid number 127 (Pro) to amino acid number 219(Gln); (d) the amino acid sequence as shown in SEQ ID NO:2 from aminoacid number 16 (Arg) to amino acid number 219 (Gin); and (e) the aminoacid sequence as shown in SEQ ID NO:2 from amino acid number 1 (Met) toamino acid number 219 (Gln).
 2. An isolated polynucleotide according toclaim 1, wherein the zsig63 polypeptide comprises a sequence of aminoacid residues selected from the group consisting of: (a) the amino acidsequence as shown in SEQ ID NO: 2 from amino acid number 16 (Arg) toamino acid number 37 (Ser); (b) the amino acid sequence as shown in SEQID NO: 2 from amino acid number 38 (Leu) to amino acid number 126 (Ala);(c) the amino acid sequence as shown in SEQ ID NO: 2 from amino acidnumber 127 (Pro) to amino acid number 219 (Gln); (d) the amino acidsequence as shown in SEQ ID NO:2 from amino acid number 16 (Arg) toamino acid number 219 (Gln); and (e) the amino acid sequence as shown inSEQ ID NO:2 from amino acid number 1 (Met) to amino acid number 219(Gln).
 3. An isolated polynucleotide according to claim 1, wherein thepolynucleotide is selected from the group consisting of: (a) apolynucleotide sequence as shown in SEQ ID NO:1 from nucleotide 173 tonucleotide 784; (b) a polynucleotide sequence as shown in SEQ ID NO:1from nucleotide 128 to nucleotide 784; and (c) a polynucleotide sequencecomplementary to (a) or (b).
 4. An isolated polynucleotide sequenceaccording to claim 1, wherein the polynucleotide comprises nucleotide 1to nucleotide 657 of SEQ ID NO:3.
 5. An expression vector comprising thefollowing operably linked elements: a transcription promoter; a DNAsegment encoding a zsig63 polypeptide comprising an amino acid sequencethat is at least 90% identical to the amino acid sequence shown in SEQID NO:2 from amino acid number 16 (Arg) to amino acid number 219 (Gln);and a transcription terminator.
 6. An expression vector according toclaim 5, further comprising a secretory signal sequence operably linkedto the DNA segment.
 7. A cultured cell into which has been introduced anexpression vector according to claim 5, wherein the cell expresses apolypeptide encoded by the DNA segment.
 8. A DNA construct encoding afusion protein, the DNA construct comprising: a first DNA segmentencoding a polypeptide selected from the group consisting of: (a) theamino acid sequence of SEQ ID NO: 2 from residue number 1 (Met) toresidue number 15 (Ala); (b) the amino acid sequence of SEQ ID NO: 2from residue number 16 (Arg) to residue number 37 (Ser).; (c) the aminoacid sequence of SEQ ID NO: 2 from residue number 38 (Leu) to residuenumber 126 (Ala); (d) the amino acid sequence of SEQ ID NO: 2 fromresidue number 127 (Pro) to residue number 219 (Gln); and (e) the aminoacid sequence of SEQ ID NO:2 from residue number 16 (Arg) to residuenumber 219 (Gln); and at least one other DNA segment encoding anadditional polypeptide, wherein the first and other DNA segments areconnected in-frame; and encode the fusion protein.
 9. A fusion proteinproduced by a method comprising: culturing a host cell into which hasbeen introduced a vector comprising the following operably linkedelements: (a) a transcriptional promoter; (b) a DNA construct encoding afusion protein according to claim 8; and (c) a transcriptionalterminator; and recovering the protein encoded by the DNA segment. 10.An isolated zsig63 polypeptide comprising a sequence of amino acidresidues that is at least 90% identical to an amino acid sequenceselected from the group consisting of: (a) the amino acid sequence asshown in SEQ ID NO: 2 from amino acid number 16 (Arg) to amino acidnumber 37 (Ser); (b) the amino acid sequence as shown in SEQ ID NO: 2from amino acid number 38 (Leu) to amino acid number 126 (Ala); (c) theamino acid sequence as shown in SEQ ID NO: 2 from amino acid number 127(Pro) to amino acid number 219 (Gln); (d) the amino acid sequence asshown in SEQ ID NO:2 from amino acid number 16 (Arg) to amino acidnumber 219 (Gln); and (e) the amino acid sequence as shown in SEQ IDNO:2 from amino acid number 1 (Met) to amino acid number 219 (Gln). 11.An isolated zsig63 polypeptide according to claim 10, wherein thepolypeptide comprises a sequence of amino acid residues selected fromthe group consisting of: (a) the amino acid sequence as shown in SEQ IDNO: 2 from amino acid number 16 (Arg) to amino acid number 37 (Ser); (b)the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number38 (Leu) to amino acid number 126 (Ala); (c) the amino acid sequence asshown in SEQ ID NO: 2 from amino acid number 127 (Pro) to amino acidnumber 219 (Gln); (d) the amino acid sequence as shown in SEQ ID NO:2from amino acid number 16 (Arg) to amino acid number 219 (Gln); and (e)the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 1(Met) to amino acid number 219 (Gln).
 12. A method of producing a zsig63polypeptide comprising: culturing a cell according to claim 7; andisolating the zsig63 polypeptide produced by the cell.
 13. A method ofdetecting, in a test sample, the presence of an antagonist of zsig63protein activity, comprising: transfecting a zsig63-responsive cell,with a reporter gene construct that is responsive to a zsig63-stimulatedcellular pathway; and producing a zsig63 polypeptide by the method ofclaim 12; and adding the zsig63 polypeptide to the cell, in the presenceand absence of a test sample; and comparing levels of response to thezsig63 polypeptide, in the presence and absence of the test sample, by abiological or biochemical assay; and determining from the comparison,the presence of the antagonist of zsig63 activity in the test sample.14. A method of detecting, in a test sample, the presence of an agonistof zsig63 protein activity, comprising: transfecting a zsig63-responsivecell, with a reporter gene construct that is responsive to azsig63-stimulated cellular pathway; and adding a test sample; andcomparing levels of response in the presence and absence of the testsample, by a biological or biochemical assay; and determining from thecomparison, the presence of the agonist of zsig63 activity in the testsample.
 15. A method of producing an antibody to zsig63 polypeptidecomprising the following steps in order: inoculating an animal with apolypeptide selected from the group consisting of: (a) a polypeptideconsisting of 9 to 204 amino acids, wherein the polypeptide consists ofa contiguous sequence of amino acids in SEQ ID NO:2 from amino acidnumber 16 (Ala) to amino acid number 219 (Gln); and (b) a polypeptideaccording to claim 10; (c) a polypeptide comprising amino acid number 16(Arg) to 37 (Ser) of SEQ ID NO:2; (d) a polypeptide comprising aminoacid number 38 (Leu) to 126 (Ala) of SEQ ID NO:2; (e) a polypeptidecomprising amino acid number 127 (Pro) to 219 (Gln) of SEQ ID NO:2; (f)a polypeptide comprising amino acid number 16 (Arg) to amino acid number219 (Gln) of SEQ ID NO:2; (g) a polypeptide comprising amino acid number1 (Met) to amino acid number 219 (Gln) of SEQ ID NO:2; (h) a polypeptidecomprising amino acid number 14 (Phe) to 19 (Arg) of SEQ ID NO:2; (i) apolypeptide comprising amino acid number 16 (Arg) to 21 (Phe) of SEQ IDNO:2; (j) a polypeptide comprising amino acid number 24 (Gly) to 29(Asp) of SEQ ID NO:2; (k) a polypeptide comprising amino acid number 25(Glu) to 30 (Asp) of SEQ ID NO:2; (l) a polypeptide comprising aminoacid number 187Glu) to 192 (Glu) of SEQ ID NO:2; (m) a polypeptidecomprising amino acid number 24 (Gly) to 33 (Pro) of SEQ ID NO:2; (n) apolypeptide comprising amino acid number 17 (Lys) to 33 (Pro) of SEQ IDNO:2; (o) a polypeptide comprising amino acid number 66 (Thr) to 73(Pro) of SEQ ID NO:2; (p) a polypeptide comprising amino acid number 103(Pro) to 108 (Gly) of SEQ ID NO:2; (q) a polypeptide comprising aminoacid number 190 (Ala) to 197 (Glu) of SEQ ID NO:2; (r) a polypeptidecomprising amino acid number 202 (Lys) to 215 (Gly) of SEQ ID NO:2; and(s) a polypeptide comprising amino acid number 190 (Ala) to 215 (Glu) ofSEQ ID NO:2; and wherein the polypeptide elicits an immune response inthe animal to produce the antibody; and isolating the antibody from theanimal.
 16. An antibody produced by the method of claim 15, which bindsto a zsig63 polypeptide.
 17. The antibody of claim 16, wherein theantibody is a monoclonal antibody.
 18. An antibody that binds to apolypeptide of claim 10.